THE ROLE OF JAZ PROTEINS IN THE REGULATION OF PLANT GROWTH AND DEFENSE By Marcelo Lattarulo Campos A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Genetics - Doctor of Philosophy 2015 ABSTRACT THE ROLE OF JAZ PROTEINS IN THE REGULATION OF PLANT GROWTH AND DEFENSE By Marcelo Lattarulo Campos When challenged with environmental stress, plants devote a significant proportion of their biosynthetic capacity to the production of secondary metabolites and other defense -related strategies. Increase d production of defensive compounds is associated with diversion of resources (e.g., carbon) from primary growth, thereby limiting plant biomass accretion. This growth -defense antagonism has a profound impact o n plant biology and ecological relationships . However, the molecular mechanisms controlling tradeoffs between growth and defense are still poorly understood. The plant signaling molecule jas monate (JA) is a key regulator of resource allocation as it reprograms transcriptional networks that appear to redirect resources from primary metabolism and growth to secondary metabolism and defense. This molecular ÒswitchÓ is mediated in part by JAZ repressor proteins that , in the absence of JA, bind to and inhibit the action of JA -related transcription factors (TFs). Stress -induced production of JA promotes the formation of COI1 -JAZ co -receptor complexes that targets JAZ proteins for rapid destructio n by the E3 ubiquitin ligase SCF COI1 , thereby rel easing TFs from inhibition. Here , I use two approaches to show that JAZ proteins have a major role in balancing resource allocation between growth and defense in the model plant Arabidopsis thaliana . First, I demonstrate that alternative splice variants of JAZ10 that are stable in the presence of high levels of JA function to attenuate JA responses , thereby prioritizing growth over defense . Second, I developed a jaz quintuple mutant (jazQ ) that lacks five of the 13 JAZ genes and show that this mutant constitutively produces defense compounds but grows slowly. The jazQ mutant was then employed as the parental line in a genetic suppressor screen aimed at uncoupling the growth -defense antagonism. Characterization of one suppressor mutant showed that loss of function of the red light receptor phytochrome B (phyB) rescues the slow growth of jazQ without significantly affecting defense traits. These findings suggest that growth -defense antagonism may not be dictated by limited metabolic resources but rather by hard -wired transcriptional programs that exert control over resource partitioning in dynamic environments. In the long term, the findings described in this dissertation may inform efforts to increase food produc tion and security while reducing the use of pesticides that are detrimental to the environment and human health. iv!ACKNOWLEDGEMENTS During my five years as a Ph.D. student at Michigan State University, I was privileged to meet wonderful people who provided me stimulus and all the support necessary to develop this research. These are some few words to thank them: I am deeply grateful to my advisor, Dr. Gregg A. Howe, for all his support, mentoring and scientific discussions thro ughout my Ph.D. studies. GreggÕs ideas, enthusiasm and encouragement during hard times were essential for me to complete this work and his leadership and serenity in conducting the lab will always serve me as an example of the scientist I want to become. I n 2009 I started to work with Gregg as a great admirer of his research and now, in 2015, I leave his lab feeling lucky and proud for all the accomplishments we achieved together. Thanks so much Gregg! I would like to express my gratitude to the members of my graduate advisory committee, Dr. Cornelius Barry, Dr. Brad Day, Dr. Sheng Yang He and Dr. Rob Last who always had the doors of their offices open to give me advices about my research and my career. Their guidance was important during my whole time here in MSU and I am in great debt with all them. I was lucky to develop my research in the 4 th floor of the Molecular Plant Sciences building, the best scientific environment I ever experienced in my life. The day -to-day interaction with many former and past members of the Howe, He and Day labs was always stimulating my scientific thoughts and giving me constant ideas on how to direct my studies. In this sense, I would like to thanks Dr. Jin -Ho Kang, Dr. Abe Koo, Dr. Marco Herde, Dr. Christine Shyu, Dr. Koich i Sugimoto, Dr. Jingling Zhai, Dr. John Withers, Bethany Huot, Dr. Alex Brutus, v!Dr. Francisco Uribe, Li Zhang, Dr. Joe (Kyaw) Aung, Alyssa Burkhardt, Dr. Katie Porter, Alex Corrion, Dr. Patricia Santos and Dr. Elizabeth Savory. Many results I obtained during my Ph.D. studies were only possible due to efforts of former Howe lab members. For this reason, I first like to thank Dr. Javier Moreno and Lalita Patel for developing the JAZ10 promoter fusion constructs and transforming som e of the jaz10 -1 lines utilized in Chapter 2 of this thesis. Besides their significant scientific contributions, Javier and Lalita are also great friends who made me laugh and enjoy my early days in the Howe lab. I also thank Dr. Yuki Yoshida for developin g many of the high order mutants described in Chapter 3 (including jazQ and jazQ phyB ), for his help in characterizing and identifying the phyB mutation in sjq11 and for all his scientific input. I am in great debt with my Brazilian comrade Dalton Oliveira , who spent hundreds of hours preparing soil trays, sowing seeds and looking at small plants to isolate the sjq and ejq lines described in Chapter 3. Finally, I would like to thank Dr. Ian Major for his friendship, his help in analyzing the RNAseq data and all his knowledge and scientific discussions. However, I will not miss your Canadian jokes, IanÉ Hahaha!! I was also lucky to share a few months working closely with three brilliant rotation students Qiang Guo, Alexandra Lantz and Brian St. Aubin and two amazing undergrads, Kayla Moses and Mirian Pimentel. Their help was vital for my research but more than that, they also showed me how much I still have to learn to become a good scientist. I have to thank to all my friend in the United States and Brazil, who provided me energy, support and tons of laughs throughout my PhD. years: Cristopher Marinos, Rom⁄rio Santos, F⁄bio Nakatani, Camila Nakatani, Dr. Kurtulus Kok, Jiying Li, Fernando Rosado, Leah Palm -Forster, Dr. Payam Mehrshahi, Dr. Sabrina Jorge, Dr. M arco Agostoni, Dr. Ipek Yapici and vi!Juliana Lopes. A special thank to my ÒbrothersÓ Vladmir Santos Jr., Pedro Nakatani and Matheus Senna who are always by my side, independent of the physical distance. None of this work would have been possible without the help of three very special friends: Cait (Gatinh ⁄) Thireault, Dr. Mieder (Viado) Palm -Forster and Dr. Andrezinho (Paolo Guerrero) Velasquez. Cait, Mieder and Andr ” were always my source of laughs and energy, the friends I always run to in search of suppor t and encouragement. You three are the main reason why I will miss the United States. Last, but not least, I dedicate this work to my girlfriend Camila Oliveira, my two sisters Fernanda and Simone and my parents Maria Teresa and Vamberto who are always by my side and are the reason why I am going back to Brazil. vii !TABLE OF CONTENTS LIST OF TABLES.. ........................................................ÉÉÉÉÉÉÉÉÉÉÉÉÉ. ix LIST OF FIGURES ........................................................ÉÉÉÉÉÉÉÉÉÉÉÉÉ. x CHAPTER ONE Literature review: Jasmonate -triggered plant immunity...................... 1 Abstract.......................................................................................................................... 2 Introduction.................................................................................................................... 3 JATI confers broad -spectrum resistance in dicots and monocots.................................. 7 Core JA signaling module.............................................................................................. 10 Activation of the core JA module.................................................................................. 16 MAMPs, HAMPs, and DAMPs ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 16 Ca2+ signaling, ROS and MAPKs ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 16 Long -distance electrical and glutamate -like receptors ÉÉÉÉÉÉÉÉÉÉÉÉ. 19 Negative regulation of JATI.......................................................................................... 20 Catabolism of JA -Ile ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 21 Stable JAZ proteins ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 22 Transcriptional JAMming ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 24 Other modes of negative regulation ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 24 Manipulation of JATI by plant -associated organisms................................................... 25 Summary and future perspectives.................................................................................. 28 Acknowledgements........................................................................................................ 32 REFERENCES.. ............................................................................................................. 33 CHAPTER TWO Alternative splicing in JAZ10 regulates jasmonate signaling in Arabidopsis thaliana ÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 56 Abstract.......................................................................................................................... 57 Introduction............................................................................................................. ....... 58 Results............................................................................................................................ 63 Expression dynamics of JAZ10 splice variants in response to woundingÉÉÉ. ......... 63 Dynamics of JAZ10 protein splice variant accumulation in response to wounding É.. 65 Stable isoforms of JAZ10 complement the JA -hypersensitive phenotype of jaz10 -1... 71 JAZ8 does not functionally complement stable isoforms of JAZ10 ÉÉÉÉÉÉÉ.. 77 Increased stability of JAZ1 0.3 is caused by alternative splicing -induced truncation of the Jas motif helix ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 80 JAZ10.3 -like JAZ genes are widespread in the plant kingdom ÉÉÉÉÉÉÉÉÉ. 86 Discussion...................................................................................................................... 88 Splice variants of JAZ10 regulat e the amplitude of JA responses.ÉÉÉÉÉÉÉÉ 88 Integrity of the JAZ degron is necessary for C OI1 interaction ÉÉÉÉÉÉÉÉÉ.. 91 Stable JAZ repressor modulate resource allocation ÉÉÉÉÉÉÉÉÉÉÉÉÉ... 93 MethodsÉÉÉÉÉ...................................................................................................... 94 Plant material and growth conditions ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 94 Wounding time -course experiment ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 94 RNA extraction and qRT -PCR ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 94 viii !Protein extraction and immunoblots analysis ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 95 Transgene constructs ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 99 Yeast two -hybrid (Y2H) analysis ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 100 In vitro pull -down assays ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 100 Site-directed mutagenesis ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 101 REFERENCES.... ........................................................................................................... 102 CHAPTER THREE Rewiring of jasmonate and phytochrome B transcriptional networks simultaneously activate plant growth and defenseÉÉÉÉ.ÉÉÉÉÉÉ 109 Abstract.......................................................................................................................... 110 Introduction............................................................................................................ ........ 111 Results............................................................................................................................ 114 The jazQ quintuple mutant shows hypersensitivity to exogenous JA and constitutive activation of defense responses ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 114 The jazQ mutation impedes plant growth ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 120 JA-response genes are constitutively upregulated in jazQ ÉÉÉÉÉÉÉÉÉÉÉ. 120 Identification of suppressors and enhancers of jaz QÉÉÉÉÉÉÉÉÉÉÉÉÉ. 124 sjq11 carries a nonsense mutation in the PHYTOCHROME B gene ÉÉÉÉÉÉÉ. 130 jazQ phyB is upregulated in growth and defense parameters ÉÉÉÉÉÉÉÉÉÉ 134 Rewiring of transcriptional networks upregulate growth and defense in jazQ phyB É. 146 Overexpression of PHYTOCHROME INTERACTING FACTOR4 in jazQ partially recapitulates the jazQ phyB phenotype ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 152 Discussion...................................................................................................................... 155 JAZ proteins inhibit defense and promote plant growth ÉÉÉÉÉÉÉÉÉÉÉÉ 156 Suppressors of jazQ identify new components involved with resource allocation decisions ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 158 Uncoupling JA and phytochrome B transcriptional networks to activate growth and defense ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 159 Methods.......................................................................................................................... 164 Plant material and growth conditions ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 164 PCR and qPCR analysis ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 165 Root growth assays ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 168 Quantification of secondary metabolites ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 168 Insect feeding assays ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 169 Rosette phenotypes and flowering time ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 169 jazQ mutagenesis experiment and identification of causal mutations in sjq plants É... 170 Global gene expression profiling (RNA -seq) ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 171 Overexpression of PIF4 in the jazQ background ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 175 Acknowledgements........................................................................................................ 176 REFERENCES.... ........................................................................................................... 177 CHAPTER FOUR Summary and future perspectives...................................................... 186 Summary of dissertation................................................................................................ 187 Future perspectives........................................................................................................ 193 REFERENCES.... ........................................................................................................... 197 ix!LIST OF TABLES Table 1.1. Examples in which there is genetic evidence for JA -mediated plant resistance to pathogens and herbivoresÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉ....É..... 9 Table 1.2. Selected examples of danger signals and effectors that modulate JA -mediated plant defense responsesÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 17 Table 2.1. Examples of JAZ genes missing the last amino acid in the JAZ degron in different plant speciesÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 87 Table 2.2. List of PCR primers used in this chapter ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 96 Table 3.1. List of selected gene onthology (GO) biological processes upregulated in jazQ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 123 Table 3.2 . List of suppressors ( sjq) and enhancers ( ejq) isolated from a M2 population of EMS -mutagenized jazQ seedsÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 125 Table 3.3. Epistatic relationships between jazQ and phyB ÉÉÉÉÉÉÉÉÉÉÉÉ.. 163 Table 3.4 . List of PCR primers used in this chapterÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉÉ 167 x! LIST OF FIGURES Figure 1.1 . Jasmonate perception by the COI1 receptor system is essential for resistance of cultivated tomato to the oomycete pathogen Pythium ÉÉ......É..ÉÉÉ. 11 Figure 1.2. Model of jasmonate -triggered plant immunity (JATI)ÉÉÉ...........ÉÉÉ.. 12 Figure 2.1. The Arabidopsis thaliana JAZ10 gene is subjected to alternative splicingÉ.. 62 Figure 2.2 . Schematic representation of the wounding experimentÉÉÉÉÉÉÉÉÉ 64 Figure 2.3. Wounding systemic expression of JAZ10 transcriptsÉÉÉÉÉÉÉ ÉÉ... 66 Figure 2.4. Quantification of JAZ10 transcripts in flowers and roots of 35S:JAZ10G transgenic plantsÉÉÉÉÉÉÉÉÉÉÉÉÉ... ÉÉÉÉÉÉÉÉÉ.... 67 Figure 2.5. Constructs used to study protein dynamics of JAZ10 splice variants in planta ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 69 Figure 2.6. Dynamics of JAZ10 splice variant accumulation in response to mechanical wounding ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 70 Figure 2.7 . Stable splice variants of JAZ10 complement the JA-hypersensitive root growth phenotype of jaz10 -1ÉÉ..É.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 73 Figure 2.8. Stable splice variants of JAZ10 complement a transcriptional phenotype of jaz10 -1.ÉÉÉÉ..ÉÉÉÉÉÉ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 75 Figure 2.9. R171A mutation disrupts JAZ10.1 -COI1 interactionÉÉÉÉÉÉÉÉÉ... 76 Figure 2.10. A stabilized form of JAZ10.1 complements the JA -hypersensitive pheno -type of jaz10 -1. ÉÉÉÉÉÉÉÉÉÉÉÉ..É..ÉÉÉÉÉÉÉÉÉÉ 78 Figure 2.11. JAZ8 does not complement the JA -hypersensitive root growth phenotype of jaz10 -1ÉÉÉÉÉÉÉÉÉÉ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 79 Figure 2.12. JAZ10.3 -COI1 interaction is restored by addition of Leu186 to the C -terminal end of JAZ10.3 ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 81 Figure 2.13. Complementation of JAZ10.3 -COI1 interaction is dependent on the physi -cal properties of amino acid (aa) residuesÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 84 Figure 2.14. JA-stability of JAZ10.3 is caused by a single amino acid truncation in the C-terminal end of its degron. ÉÉÉ......ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 85 xi! Figure 3.1. The jaz quintuple (jazQ) mutant is defective in JAZ1, JAZ3, JAZ4, JAZ9 and JAZ10 ÉÉÉÉÉÉÉÉÉ...ÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉ... 115 Figure 3.2. jazQ is hypersensitive to exogenous JAÉÉÉÉ ÉÉÉÉÉÉÉ...ÉÉÉ 117 Figure 3.3. Constitutive accumulation of secondary metabolites in jazQ .É...ÉÉÉÉ... 118 Figure 3.4 jazQ exhibits increased resistance to insect herbivory. ÉÉÉÉÉ...ÉÉÉ. 119 Figure 3.5. Growth processes are hindered in jazQ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 121 Figure 3.6 . Genes involved in glucosinolate biosynthesis and breakdown are upregu -lated in jazQ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...ÉÉÉ. 128 Figure 3.7 . Isolation of enhancers and suppressor mutants from a population of EMS -mutagenized jazQ M2 plants .ÉÉÉ..ÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉ.. 129 Figure 3.8. sjq66 carries a mutation in the CORONATINE INSENSITIVE1 gene ÉÉÉ.. 131 Figure 3.9. sjq11 suppresses the slow -growth phenotype of jazQ but not its anthocyanin accumulation in petioles ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 132 Figure 3.10. sjq11 shows improved growth and retains hypersensitivity to JA treatment . 133 Figure 3.11. sjq11 seedlings develop long hypocotyls under white lightÉÉÉÉÉÉ... 135 Figure 3.12 . sjq11 is impaired in red light perceptionÉ ÉÉÉÉÉÉÉÉÉÉÉÉÉ 136 Figure 3.13. sjq11 harbors a mutation in the PHYTOCHROME B (phyB ) gene ÉÉÉÉ. 137 Figure 3.14. Genetic non -complementation of log hypocotyl phenotype in sjq11 and phyB -9.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.... 138 Figure 3.15. jazQ phyB plants combine the stronger anthocyanin accumulation of jazQ with the large rosette size of phyB -9ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 139 Figure 3.16. Growth parameters are improved in jazQ phyB ÉÉÉÉÉÉÉÉÉÉÉ.. 140 Figure 3.17. jazQ phyB is hypersensitive to JA and accumulates more glucosinolates than WTÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 143 Figure 3.18. jazQ phyB is more resistant to insect herbivoryÉÉÉÉÉÉÉÉÉÉÉ. 144 Figure 3.19. phyB -9 plants are extremely susceptible to insect herbivoryÉÉÉÉÉÉ.. 145 xii !Figure 3.20. The combination of phyB -9 and jazQ leads to additive transcriptional ef -fects in jazQ phyB ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 147 Figure 3.21. Genes associated with glucosinolate biosynthesis are upregulated in jazQ and jazQ phyB but partially downregulated in phyB -9.ÉÉÉ...ÉÉÉÉÉ. 148 Figure 3.22. The combination of phyB -9 and jazQ leads to additive and synergistic transcriptional reprogramming in jazQ phyB ÉÉÉÉ...ÉÉÉÉÉÉÉ... 150 Figure 3.23. Overexpression of PIF4 in jazQ leads to partial rescue of growth without affecting defenseÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 153 Figure 3.24. Overexpression of PIF4 in jazQ does compromise resistance to insect herbivoryÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 154 Figure 3.25. Removal of transcriptional regulators rewires a regulatory network to al -low concomitant activation of growth and defense ÉÉÉÉÉÉÉÉÉ... 161 Figure 3.26. Comparison of gene expression profiles between wild type (WT) and various mutants analyzed in this studyÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 173 Figure 3.27. Validation of RNA -seq data by qPCRÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 174 Figure 4.1. JAZ proteins are modulators of plant growth and defenseÉÉÉÉÉÉÉ... 192 1! CHAPTER ONE Literature review: Jasmonate -triggered plant immunity Work presented in this chapter has been published: Campos ML, Kang JH and Howe GA. (2014). Jasmonate -triggered plant immunity. J Chem Ecol 40:657-675 2!Abstract The plant hormone jasmonate (JA) exerts exquisite control over the production of chemical defense compounds that confer resistance to a remarkable spectrum of plant -associated organisms, ranging from microbial pathogens to vertebrate herbivores. The underlying mechanism of JA -triggered immunity (JATI) can be conceptualized as a multi -stage signal transduction cascade involving: i) pattern recognition receptors (PRRs) that couple the perception of danger signals to rapid synthesis of bioactive JA; ii) an evolutionarily conserved JA signaling module that links fluctuating JA levels to changes in the abundance of transcriptional repressor proteins; and iii) activation (de -repression ) of transcription factors that orchestrate the expression of myriad chemical and morphological defense traits . Multiple negative feedback loops act in concert to restrain the duration and amplitude of defense responses, presumably to mitigate the metaboli c cost and negative impact of JATI on plant fitness. The convergence of diverse plant - and non -plant -derived signals on the core JA module indicates that JATI is a general response to perceived danger. However, the modular structure of JATI may accommodate attacker -specific immune responses through evolutionary innovation of PRRs (inputs) and chemical defense traits (outputs). The efficacy of JATI as a defense strategy is highlighted by its capacity to shape natural populations of plant attackers, as well a s the propensity of plant -associated organisms to subvert or otherwise manipulate JA signaling. As both a cellular hub for integrating informational cues from the environment and a common target of pathogen effectors , the core JA module provides a focal point for understanding immune sys tem networks and the evolution of chemical diversity t hroughout the plant kingdom. 3!Introduction Plants are a source of nutrition for a vast biota in terrestrial environments. Selective pressure imposed by pathog ens and herbivores has s haped the evolution of an astonishing array of specialized plant defense compounds that exert direct toxic, anti -nutritional , or repellant effects on plant consumers. Other defensive compounds work indirectly by attracting natural enemies of plant -associated organisms . Strategies to deploy these protective chemical shields and associated morphological structures may be constitutive or inducible . It is thought that n atural selection , at least in some plant species, favor ed the evolut ion of induced defenses because they have lower resource allocation costs than constitutive resistance traits (Baldwin 1998; Herms and Mattson 1992; Thaler et al. 2012). A key feature of many induced defense traits is their expression in tissues distal to the site of infection or attack . The combined effect of local and systemic defense res ponses provide s broad -spectrum resistance agains t subsequent biotic attacks , and constitute s a form of induced immunity (Fu and Dong 2013; Howe and Jander 2008; Jones an d Dangl 2006 ). Intensive r esearch efforts to understand the molecular mechanisms and evolution ary ecolog y of induced immunity have focused on the question of how plants recognize a foreign threat . Significant insight into th is question has come from molecular genetic analyses of plant -pathogen interactions . P attern -triggered immunity (PTI) confers basal resistance and is mediated by cell surface -localized pattern recognition receptors (PRRs) that bind conserved foreign molecul es, known collectively as microbial/ pathogen -associated molecular patterns (MAMPs) (Chisholm et al. 2006; Dodds and Rathjen 2010 ; Jones and Dangl 2006 ). A second layer of induced resistance , referred to as effector -triggered immunity (ETI), r elies on polymorphic intracellular resistance (R) proteins to detect effector molecules that plant attackers deliver into host cells to counteract defense. ETI responses often include localized host cell death and are 4!qualitatively similar , though typically more robust and faster , than PTI responses (Dodds and Rathjen 2010) . Major conceptual contribution s of the PTI/ETI paradigm include the distinction between plant defense responses trigg ered by conserved patterns versus effectors , and a model of how these branches of immunity influence the evolution of plant -pathogen associations (Jones and Dangl 2006) . The PTI/ETI model has also influenced current views of how plants recognize attack by arthropod herbivores, which constitute the majority of plant -consuming species on Earth (Erb et al. 2012 ). Accordingly, eliciting compounds produced by arthropod herbivores have been dubbed herbivore -associated molecular patterns (HAMPs) ( Felton and Tumlison 2008; Mithofer and Boland 2008 ). In addition to cell surveillance systems that recognize foreign threats in the form of MAMPs/HAMPs and effectors, it has long been known that plant -derived ( i.e., self) signals are also potent elicitors of local and systemic defense responses ( Bergey et al. 1996; Green a nd Ryan 1972; Heil et al. 2012; Huffaker et al. 2006 , 2011; Krol et al. 2010; Mousavi et al. 2013). These endogenous elicitors are produced in res ponse to general cellular injury and may be classified as damage -associated molecular patterns (DAMPs) . Becaus e DAMPs are generated in response to diverse types of tissue injury, their role in cellular recognition of pathogen attack has traditionally been ignored . However, the recent identification of DAMP receptors and associated signal transduction components (Brutus et al. 2010; Choi et al 2014; Mousavi et al., 2013; Yamaguchi et al . 2006 , 2010 ) is shaping a broader view of how plant cells perceive and respond to injurious threats (Boller and Felix 2009; De Lorenzo et al. 2011; Heil 2009; Koo and Howe, 2009). T he diversity of conserved patterns that trigger local and systemic defense re actions supports the concept that cellular perception of Òdanger Ó, regardless of its source, is a unifying 5!principle of induced immunity in plants and animals (Boller and Felix 2009; Koo and Howe 2009; Lotze et al. 2007; Matzinger 2002 ). A second major question surrounding induced immunity concerns the extent to which cellular recognition of a threat is translated into a host response that neutralizes the attacking pathogen or herbivore . Indeed, g enome -wide t ranscriptom e studies indicate a significant degree of overlap in molecular responses triggered by different MAMPs/HAMPs/DAMPs and effectors (Bidart -Bouzat and Kliebenstein 2011; Caillaud et al. 2013; Gouhier -Darimont et al. 2013; Kim et al. 2014; Navarro et al. 2004; Reymond et al. 2004 ; Reymond et al. 2004; Tao et al. 2003; Thilmony et al. 2006; Tsuda et al. 2008, 2009; Wise et al. 2007; Zhuro v et al. 2014 ). There is also evidence to indicate that PTI and ETI converge on similar downstream signaling components, including MAP kinase pathways, ROS production, and calcium -dependent signaling events (Romeis and Herde 2014 ; Sato et al. 2010 ). Althou gh quantitative differences in the timing and strength of induction is likely to shape the outcome of specific plant -attacker associations (De Vos et al. 2005; Katagiri and Tsuda 2010; Tao et al. 2003; Wise et al. 2007 ), most evidence indicates that specific danger signals trigger general host defense responses that are effective against broad classes of pathogens and herbivores (Erb et al. 2012). The central role of small -molecule hormones in controlling the expression of chemical and morphological defense traits provides a n impetus for describing induced immun ity from the perspective of phytohormone networks (Erb et al. 2012; Pieterse et al. 2009; Reymond and Farmer 1998 ). It is now evident that diverse danger signals converge on the immune -promoting effects of two major defense hormones, jasmon ic acid (JA) and salicyl ic acid (SA) . A wealth of genetic evidence indicates that J A-triggered immunity (JATI) and SA -triggered immunity (SATI) contribute to plant resistance to many, if not most, pathogens and arthropod herbivores 6!studied to date . JATI and SATI interact and further crosstalk with other plant hormone pathways to activate the most effective responses to the particular type of attacking organism, in a way that finel y balance tradeoffs between defense , growth, and reproductive success. Here, we focus on recent advances in understanding how JA and its cognate receptor system control a bewildering array of defense responses across the plant kingdom . We describe JATI as a multistage process in which a highly conserved core JA module links a variety of PRR -based recognition systems (inputs) to the expression of specific defense traits (outputs). Based on these considerations, we propose that the regulatory structure of JAT I has potential to create new specificities of host resistance through evolutionary innovation of input and output modules. We also highlight the various ways in which plant -associated organisms manipulate JATI to their own advantage. These new mechanistic insights will help to explain how JATI shapes patterns of chemical diversity and species interaction in the plant kingdom, and how these relationships affect genome evolution to modulate phenotypic plasticity. Our focus on JA is not intended t o minimize the role of other signals in coordinating plant defense responses , or to distract from the important endeavor of understanding the complexities of phytohormone networks and their relationship to induced immunity (Ballar” 2014; Kazan and Manners 2012; Kim et al. 2014; Mukhtar et al. 2011; Pieterse et al . 2009;) . Nevertheless, we subscribe to the view that an accurate, and ultimately predictive, understanding of interconnected signaling networks depends on knowledge of how individual signals are produced and perceived at the molecular level . Readers a re referred to several excellent review articles for a comprehensive discussion of JA -mediated signal transduction , its interaction with other signaling pathways , and the function of JA in develo pment processes (Ballar” 2011; Browse 209; Huot et al., 2014; Kazan and Manners 2008, 7!2013; Kombrink 2012; Meldau et al. 2012; Moreno and Ballar” 2014; Pauwels and Goossens 2011; Robert -Seilianiantz et al. 2011; Shyu and Brutnell 2015; Wasternack and Hause 2013) JATI confers broad -spectrum resistance in dicots and monocots The central role of JA as an activating signal for induced immunity is grounded in three general observations: First, biotic attack and other forms of tissue injury result in the rapid synthesis of JA and its receptor -active derivative, jasmonoyl -L-isoleucine (JA-Ile). Stress -induced accumulation of JA -Ile occurs in both above - and below -ground tissues and, depending on the eliciting signal and tissue type , is a systemic response (Chauvi n et al. 2013; Fragoso et al. 2014; Grebner et al. 2013; Koo et al. 2009; Mousavi et al. 2013; S chilm iller and Howe 2005 ). Second, JA promotes the expression of virtually all major classes of secondary metabolites and proteins that have established roles in defense , including alkaloids, terpenoids, phenylpropanoids, amino acid derivatives, anti -nutritional proteins, and some pathogenesis -related (PR) proteins (Browse and Howe 2008; De Geyter et al. 2012; De Vleesschauwer et al. 2013; Farmer and Ryan 1990; Gonzales -Vigil et al. 2011; Mohan et al. 2006; Van Loo n et al. 2006) . The JA pathway also promot es the development of morphological s tructures , including glandular trichomes , resin ducts, and n ectaries that produce a rich variety of compounds servin g direct and indirect roles in defense (Dicke and Baldwin 2010; Hudgins et al. 2004; Li et al. 2004; Peiffer et al. 2009; Qi et al. 2011; Radhika et al. 2010; Traw and Bergelson 2003; Van Poecke and Dicke 2002; Yoshida et al. 2009). Finally, studies employing JA mutants have demonstrated the crucial role of this hormone in plant protection against diverse biota (Browse and Howe 2008) . A mong the plant -associated organisms whose fitness is curtailed by JATI are necrotrophic and (hemi)biotrophic p athogens, mutualistic fungi, nemat odes, leaf hopper s, beetles, caterpillars , thrips, spider mites , f ungus gnats, 8!slugs , crustaceans, and some vert ebrate herbivores (Table 1.1). Indeed, it is reasonable to think that the number of plant -eating species affected by JATI may exceed the total number of plant species on Earth . Much of our knowledge about the protective effects of JATI comes from studies on a limited number of dicot species, including Arabi dopsis, tomato, and tobacco. These studies have led to the generalization that tissue -consuming insect herbivor es and necrotrophic pathogens are particularly sensitive to JATI , whereas biotrophic organisms that obtain nutrients from living host tissues succumb to the effects of SATI (Cailldaud et al. 2013; Glazebrook 2005 ; Pieterse et al. 2009 ). There is every indication that JA promotes resistance of monocot and gymnosperm species to a wide range of pathogens and herbivores (Hudgins et al. 2004; Schmelz et al. 2013; Shyu and Brutnell 2015; Yan et al. 2012 ; Ye et al. 2012; Zulak and Bohlmann 2010 ). In contrast to the biotroph /necrotroph dichotomy that has emerged from studies with Arabidopsis , it is noteworthy that JA is required for induced immunity of rice to (hemi)b iotrophi c parasites , including t he root knot nematode ( Meloidogyne graminicola) and Xanthomonas oryzae (Nahar et al. 2011 ; De Vleesschauwer et al. 2013 ). This conclusion is consistent with the ability of JA to elicit expression of many PR genes and defensive secondary metabolites in rice and maize (Mitsuhara et al. 2008 ; Schmelz et al. 2011; Yamane 2013 ). Given t hese findings , together with the high endogenous levels of free SA in rice leaves (Silverman et al. 1995), the precise role of SA as a signal for induced immunity in monocots awaits for further clarification (De Vleesschauwer et al. 2013). The serendipitous discover y that JA mutants maintained in artificial growth environments succumb to attack by unsuspected pathogens and herbivores vividly demonstrates the robust protection afforded by JATI (Browse and Howe 2008). Elegant field studies have em- 9!Table 1.1. Examples in which there is genetic evidence for JA -mediated plant resistance to pathogens an d herbivores. ORGANISM HOST PLANT REFERENCE Pathogenic bacteria Erwinia carotovora Xanthomonas oryzae Arabidopsis thaliana (Brassicaceae) Oryza sativa (Poaceae) Norman -Setterblad et al. 2000 Yamada et al. 2012 Necrotrophic fungi / oomycetes Alternaria brassicicola Botrytis cinerea Pythium spp A. thaliana (Brassicaceae) Zea mays (Poaceae) Solanum lycopersicum (Solanaceae) Thomma et al. 1998 Vijayan et al. 1998 Staswick et al. 1998; Yan et al. 2012 This study Nematodes Meloidogyne graminicola O. sativa (Poaceae) Nahar et al. 2011 Mollusks Arion lusitanicus A. thaliana (Brassicaceae) Falk et al. 2013 Crustaceans Porcellio scaber Armadillidium vulgare A. thaliana (Brassicaceae) O. sativa (Poaceae) Farmer and Dubugnon 2009 Cell content feeders Tetranychus urticae (Acari) Frankliniella occidentalis (Thysanoptera) S. lycopersicum (Solanaceae) A. thaliana (Brassicaceae) Solanum lycopersicum (Solanaceae) A. thaliana (Brassicaceae) Li et al. 2004 Zhurov et al. 2014 Li et al. 2002 Abe et al. 2009 Piercing -sucking insects Myzus persicae (Hemiptera) Empoasca sp. (Hemiptera) A. thaliana (Brassicaceae) Nicotiana attenuata (Solanaceae) Ellis et al. 2002 Kessler et al. 2004 Leafminer insects Scaptomyza flava (Diptera) A. thaliana (Brassicaceae) Whiteman et al. 2011 Leaf / root chewing insects Manduca sexta (Lepidoptera) Spodoptera frugiperda (Lepidoptera) Bradysia impatiens (Diptera) Spodoptera exigua (Lepidoptera) N. attenuata (Solanaceae) S. lycopersicum (Solanaceae) A. thaliana (Brassicaceae) Zea mays (Poaceae) Howe et al. 1996; Kessler et al. 2004; Campos et al. 2009 McConn et al. 1997 Yan et al. 2012 Vertebrate herbivores Eurotestudo boettgeri A. thaliana (Brassicaceae) Mafli et al. 2012 10! ployed natural and synthetic genetic variation to demonstrate this phenomenon in natural habitats and have established the ecological importance of JATI in shaping herbivore community composition (Kallenbach et al. 2012; Kessler et al. 2004; Thaler et al. 2001; Zt et al. 2012 ). We employed this unbiased Òask the plantÓ approach to query the biological role of the JATI in mediating interaction of cu ltivated tomato ( S. lycopersicum) with potential biotic attackers in the field. Previous studies at this field site showed that glandular trichomes, whose development on tomato leaves controlled in part by the JA pathway ( Boughton et al 2005; Li et al 2004; Peiffer et al 2009 ), provide an important layer of anti -insect defense (Kang et al 20 10a, 2010b , 2014). Rep licated field trials showed that a tomato mutant ( jai1 -1) lacking the JA -Ile receptor suffered 100% mortality from root rot disease caused by the oomycete pathogen Pythium (Figure 1.1). Similar re sults have been reported for JA mutants of Arabidopsis and maize ( Staswick et al 1998; Vijayan et al 1998; Yan et al. 2012 ). These collective studies provide a compelling demonstrat ion of the efficacy of JATI in protecting diverse plant s against the same soil -borne pathogen. Core JA signaling module The signal transduction events that couple perception of danger signals at the cell surface to the expression of JA-responsive defense genes relies on a n evolutionarily conserved core apparatus to synthesize and perce ive JA -Ile (Fig ure 1 .2) (Chico et al . 2008 ; Katsi r et al. 2008a ). A crucial feature of JA -Ile as a trigger for defense gene expression is its rapid and reversible accumulation in vegetative tissues that are frequently targeted for attack. Unstressed leaves of Arabidopsis , for example, contain extremely low or undetectable amounts of bioactive JA (Gla user et al. 2008; Koo and Howe 2009). J A synthesis is initiated in plastids from the pre-existing C18 precursor li- 11! Figure 1.1 . Jasmonate perception by the COI1 receptor syst em is essential for resistance of cultivated tomato to the oomycete pathogen Pythium. (A and B) Wild-type (cv Castlemart) and (C and D) jai1 -1 mutant plants grown for three weeks in a growth chamber without visible signs of disease were transplanted to a field plot at Michigan State University, East Lansing, MI. Two weeks after transplanting, all jai1 -1 plants (n = 30) died from a disease that w as diagnosed as Pythium stem/root rot by the MSU Diagnostics Lab. Sequencing of PCR products derived from 5.8S ribosomal genes and internal transcribed spacer region in infected tomato tissue confirmed the presence of Pythium ultimum . Of several hundred wild-type ( Jai1 /Jai1 ) plants grown side -by-side at the same field site, none showed symptoms of the disease. The figure shows photographs of representative wild -type and jai1 -1 mutant plants two weeks after transplantation. Identical results were obtained in three independent trials performed at the same site. 12! Figure 1.2. Model of jasmonate -triggered plant immunity (JATI). Danger signals (MAMPs/HAMPs) derived from attacking organisms and damaged plant cells (DAMPs) are recognized by pattern recogni tion receptors (PRRs) at the cell surface. PRR activation is coupled to intracellular signaling systems involving MAP kinase pathways (MAPK), Calcium ion -sensing proteins, and reactive oxygen species (ROS), among others. How these signaling events are conn ected to activation of the core JA signal module, which includes JA biosynthesis from its precursor linolenic acid (LA), is largely unknown (?). Plastidic and peroxisomal enzymes convert LA to jasmonic acid (JA), which is the substrate for synthesis of JA-Ile in the cytosol. Within the nucleus, JA -Ile promotes JAZ -COI1 interaction and targets JAZs for proteolytic degradation by the ubiquitin -proteasome system. Removal of JAZ alleviates TFs from repression, thereby activating the expression of JA -responsive genes and the production of defense -related chemicals and morphological structures (Defense). Several mechanisms to attenuate signaling through the core module have been elucidated, including catabolism of JA -Ile via !-oxidation and hydrolysis, de novo syn thesis of JAZ repressors that are stable in the presence of JA -Ile, and accumulation of JAM TFs that negatively regulate transcription. Pathogen -derived effectors target the core JA signal module to disrupt hormonal balance and induced immune responses. Ab breviations: Microbe -associated molecular patterns 13! Figure 1.2 (contÕd). (MAMPs); Herbivore -associated molecular patterns (HAMPs); Damage -associated molecular patterns (DAMPs), Mitogen -activated protein kinase (MAPK); Reactive oxygen species (ROS), 12 -oxo-phytodienoic acid (OPDA), "-oxidation ( "-ox), jasmonoyl -L-isoleucine (JA -Ile), JASMONATE -ZIM domain (JAZ), JA -related transcription factor (TF), JASMONATE-ASSOCIATED MYC2 -LIKE (JAM), 12 -carboxy -JA-Ile (12COOH -JA-Ile). 14! nolenic acid (LA) . LA is convert ed in the plastid to a cyclic 12-oxo-phytodienoic acid (OPDA) intermediate , which is then transported to the peroxisome for subsequent reduction and !-oxidation steps that give rise to JA (Howe and Schilmiller 2002; Schaller and Stintzi 2009; Wasternack and Hause 2013). JA is conjugated to Ile in the cytosol to produce JA -Ile (Kang et al. 2006; Staswick and Tiryaki 2004). As the receptor -active form of the hormone (Fonseca et al. 2009; Katsir et al. 2008b; Sheard et al. 2010; Staswick 2004; Thines et al . 2007 ), JA -Ile presumably diffuses into the nucleus where it is perceived by its receptor. Many genes required for JA -Ile biosynthesis are coordinately upregulated by the J A signaling pathway ( Browse 2009; Koo et al. 2006; Wasternack and Hause 2013). Although this observation suggests a positive feedback loop to amplify JA responses, the existence of JA-inducible negative feedback loops (see below) highlights the complexity of processes involved in JA -Ile homeostasis. JA-Ile controls defense gene expression by promoting the de struction of JAZ (JAsmonate ZIM -domain) transcriptional repressors via the ubiquitin -26S proteasome system (Chini et al. 2007; Thines et al. 2007; Yan et al. 2007 ). JAZ proteins are defined by two highly conserved sequence motifs referred to as ZIM (or TIFY ) and Jas ( Browse 2009 ; Chung et al. 2009 ; Yan et al. 2007 ). In the absence of stress, low level s of JA -Ile permit JAZ prot eins bind to and repress TFs in the nucleus. The basic helix -loop -helix TF MYC2 an d its closely related paralogs MYC3 and MY C4 are the most extensively studied JAZ -interacting TFs having a direct role in JATI (Fern⁄ndez -Calvo et al. 2011; Kazan and Manners 2013 ; Schweizer et al. 2013 ). Nuclear localization of JAZ repressors is dependent of physical association with MYC2 (Withers et al. 2012). Within the nucleus, t he repressive function of some JAZ requires the NINJA (Novel Interactor of JAZ) protein to mediate interaction with corepres sor s such as TOPLESS (TPL) (Pauwels et al 2010; Acosta et al 2013) . NINJA contains a so -called EAR (ERF -associated 15!amphiphilic repression) motif that binds TPL and TPL -related corepressors (Pauwels et al. 2010; Szemenye i et al. 2008 ). Other JAZ protein s, (e.g. , JAZ8 ), contain an EAR motif to allow direct recruit ment of TPL and repression of JA responses independently of NINJA (Shyu et al . 2012 ). A rapidly expanding list of JAZ - and MYC2 -interacting regulatory proteins that participate in other hormone -response pathways indicate that the JAZ -TF interactome is a cellular hub for integrating diverse environmental and developme ntal signals ( Ballar” 2014; Hou et al. 2010; Hu et al. 2013a; Kazan and Manners 2013; Nakata et al. 2013; Pauwels et al. 2010; Qi et al. 2011 , 2014; Song et al. 2011 , 2014; Toda et al. 2013; Yang et al. 2012b; Zhu et al. 2011 ). In response to perception of signals that trigger JA -Ile synthesis , JA -Ile promotes direct binding of JAZ repressors to the F -box protein COI1 (CORONATINE INSENSITIVE1), which is the specificity determinant of the E3 ubiquitin ligase SCF COI1 (Katsir et al. 2008b; Melotto et al. 2008; Sheard et al. 2010; Thines et al. 2007; Xie et al. 1998 ). U biquitin -dependent degradation of JAZ prot eins relieves repression of TFs , thereby allowing the expression of JA -response genes (Fig ure 1.2). The timing, amplitude, and duration of JA responses appears to be is controlled primarily by the intracellular level s of JA -Ile ( Koo et al., 2009; Wasternack and Hause 2013). Moreover, t he speed with which danger signals are transmitted to gene activation via the core JA pathway may be remarkably fast. Crush -type wounds inflicted to Arabidopsis leaves , for example, result in increased JA-Ile levels with in minute s of tissue damage, with increased accumulation of primary JA -res ponse transcripts observed within 5 min of wounding (Chauvin et al. 2013; Chung et al. 2008 ). Mechanical tissue damage also triggers rapid systemic responses, including JA -Ile accumulation, d egradation of JAZ proteins, activation of JA -response genes , and induced r esistance (Acosta et al 2013; Green and Ryan 1972; Koo et al. 2009; Mousavi et al. 2013; Zhang and Turner 2008 ). 16!Activation of the core JA module MAMPs, HAMPs, and DAMPs Ð Available evidences indicate that JA/JA -Ile synthesis is controlled at the post -transcriptional level via activation of pre -existing JA biosynthetic enzymes (Wasternack and Hause 2013). Although the precise mechanism of activation remains to be determined, a wide range of endogenous (DAMPS) and foreign (MA MP/HAMP) signals have been implicated in the process (Fig ure 1 .2; Table 1.2). Analysis of phytohormone production and defense gene expression in response to elicitors such as flagellin, elongation factor -Tu (EF -Tu) , and chitin , for which the cognate PRRs have been identified, ind icate that these conserved bacterial and fungal patterns activate multiple branches of induced immunity, including JATI (Kim et al. 2014) . Several HAMPs, including fatty acid -amino acid conjugates (FACs) , also amplify JA responses (McCloud and Baldwin 1 997; Sch melz et al. 2003, 2007 ). Elicitation of JA-mediated defense responses by DAMPs, including the 18 -amino -acid peptide systemin and cell wall -derived oligogalacturonides (OGs) , is consistent with the ability of these compounds to stimulate JA synthesis (Doares et al. 1995; Lee and Howe 2003). Likewise, endogenous peptide elicitors from Arabidopsis (AtPep1) and maize (ZmPep3) exert potent stimulatory effects on JATI (Table 1.2) ( Huffaker et al. 2006, 2013). Identification of plant receptors for OGs and AtPep1 marks a major advance in effort s to understand the contribution of DAMPs to plant immunity (De Lorenzo et al., 2011; Krol et al., 2010; Yamaguchi et al., 2006;) . Equally exciting is the recent discovery of the receptor for extracellular ATP, which exhibits properties of a danger signal released by damaged cells ( Choi et al. 2014 ; Song et al. 2006 ). Ca2+ signaling, ROS and MAPKs - It is generally accepted that JA synthesis is initiated in the plastid by stress -induced activation of lipases that release fatty acid precursors of JA (Bergey et 17!Table 1.2. Selected examples of danger signals and effectors that modulate JA -mediated plant defense responses. SIGNAL MECHANISM OF PERCEPTION / ACTION REFERENCES DAMPs AtPep1 LRR -RK receptors PEPR1 and PEPR2. Activates JA - and SA -dependent innate immune responses. Huffa ker et al. 2006; Yamaguchi et al. 2006 Systemin Receptor unknown (presumed LRR -RK). Elicits JA synthesis and production of defense compounds. Pearce et al. 1991 ZmPep3 Unknown receptor. Activates JA synthesis and production of defense compounds. Huffaker et al. 2013 Oligogalacturonides WAK1 receptor. Activates JA synthesis and production of defense compounds. Doares et al. 1995 Brutus et al. 2010 Extracellular ATP DORN1 receptor. Activates transcriptional responses that are similar to wound responses. Choi et al. 2014 MAMPs/HAMPs Flagellin (bacterial pathogens) LRR -RK receptor FLS2. Activates the JA and other branches of induced immunity. Chinchilla et al. 2006; Kim et al. 2014 Elongation factor -Tu (bacterial pathogens) LRR -RK receptor EFR. Activates the JA and other sectors of induced immunity. Zipfel et al. 2006; Kim et al. 2014 Chitin (fungal patho gens) LysM -RK receptor CERK1. Predominately activates the JA sector of induced immunity. Wan et al. 2008; Kim et al. 2014 Volictin and other fatty acid -amino acid conjugates (Lepidopteran herbivores) Unknown receptor. Released from insect oral secretions to stimulate JATI. Alborn et al. 1997; Halitschke et al. 2001 Inceptin (Lepidopteran herbivores) Unknown receptor. Activates JA accumulation and associated defense responses. Schmelz et al. 2007 Physical signals Electrical potentials (Mechanical tissue damage) Glutamate -like receptors mediate systemic JA responses. Mousavi et al. 2013 Microbial effectors Coronatine (Pseudomonas syringae ) JA-Ile analog that promotes formation of COI -JAZ co -receptor complexes and JAZ degradation. Katsir et al. 2008b; Sheard et al. 2010 HopZ1a (Pseudomonas syringae ) Putative acetytransferase that promotes COI1 -dependent JAZ degradation. Jiang et al. 2013 HopX1 (Pseudomonas syringae ) A cysteine protease that promotes COI1 -independent JAZ degradation. Gimenez -Ibanez et al. 2014 MiSSP7 (Laccaria bicolor ) An effector from a mutualistic fungus that binds to and protects JAZ6 from JA/COI1 -induced degradation. Plett et al. 2014b HaRxL44 (Downy mildew) Promotes degradation of Mediator subunit 19a to activate JA responses and suppress SATI. Caillaud et al. 2013 18! al. 1996; Hyun et al. 2008; Wasternack and Hause 2013 ). Alternatively, there is evidence to suggest that tissu e damage may stimulate JA synthesis from an existin g pool of OPDA (Koo et al. 2009). Regardless of the precise mechanism involved, a major gap in our understanding of JATI concerns the molecular events that link perception of MAMP/HAMP /DAMPs by PRRs to accumulation of JA -Ile (Fig ure 1.2). Among the intracellular signals implicat ed in this process are calcium ions , reactive oxygen species (ROS) , and mitogen -activated protein (MAP) kinase cascades . Calcium ions have l ong been recognized as ubiquitous second messengers in signal transduction pathways. The involvement of Ca 2+ in JATI is supported b y studies showing that cytosolic Ca 2+ levels increase in response to herbivore feeding and treatment with exogenous MAMP/HAMP/DAMPs (Arimura and Maffei 2010; Jeter et al. 2004; M affei et al. 2004, 2006 ). Changes in membrane polarizati on caused by wounding and insect attack also increase the level of cytosolic Ca 2+ (Maffei et al. 2006) . Ca2+ fluxes and associated Ca 2+-binding proteins, including calmodulin and Ca 2+-dependent protein kinases (CDPKs), exert control during t he activation of JA-response genes (Bonaventure et al. 200 7; Boudsocq et al. 2010; Levy et al. 2005; R omeis and Herde 2014 ; Yang et al. 2012a ). Dynamic changes in c ytosolic Ca 2+ levels during plant -attacker interactions are tightly linked to the production of reactive oxygen species (ROS), including hydrogen peroxide (Arimura and Maffei 2010) . Alterations in cell ular redox status are associated with local and systemic JATI , and have been li nked to the activity of the respirato ry burst oxidase homolog D (RBOH D) (Miller et al. 2009 ; Orozco -C⁄rdenas et al. 2001 ). Direct phosphorylation of RBOH D by the PRR -associated kinase BIK1 provides a mechanism to integrate MAMP perception with calcium -based regulation of immune function (Kadota et al. 2014; Li et al. 2014). 19!MAP kinase signaling cascades serve a prominent role in the early steps of induced immunity ( Asai et al. 2002; Schweighofer et al. 20 07; Seo et al. 2007; Wu and Baldwin 2010 ; Zhang and Klessig 2001 ). Plants silenced in the expression of specific MAPKs showed reduced JA biosynthesis and decreased expression of JA -related defense genes , suggesting that these kinases control an early step in the activation of JA synthesis (Kandoth et al. 2007 ; Wu et al. 2007). The manner in which MAPK cascades are linked to a specific step in the JA biosynthesis pathway, however, remains unknown. Long-distance electrical and glutamate -like receptors - One of the most fascinating and least understood areas of plant signaling concerns the mechanism by which mechanical tissue injury , including that elicited by chewing insects, results in rapid systemic changes in defense gene expression (Koo and Howe 2009) . Studies in Arabidopsis , for example, show that a signal generated at the site of leaf injury travels rapidly (2 -3 cm/min) to trigger JA -Ile synthesis and associated JA responses in undamaged leaves (Glauser et al. 2008; Koo et al. 2009) . Despite the importance and wide -spread occurrence of this phenomenon, the molecular and genetic basis of rapid systemic JATI signaling has being uncertain . A recent study by Mousavi et al. (2013) built on previous work showing that tissue damage results in cha nges in electrical activity and membrane depolarization, which are associated with activation of JA responses in systemic tissues (Mousavi et al., 2013; Wildon et al. 1992; Zimmermann et al. 2009 ). A screen for mutants that exhibit reduced wound -trigge red changes in eletric potential showed that members of the GLUTAMATE RECEPTOR -LIKE (GLR) family of ion channel proteins are required for the response; glr3.3 and glr3.6 mutants are deficient in electrical activity in wounded leaves and show reduced expres sion of JA -responsive genes in distal undamaged leaves (Mousavi et al. 20!2013). Systemic depolarization events were triggered in a GLR3.3/GRL3.6 -dependent manner by caterpillar feeding but not by caterpillar walking in the leaf (Mousavi et al. 2013; Salvador -Recatal ‹ et al. 2014). The ability of mechanical leaf wounding and insect chewing to elicit comparable changes in electrical activity indicates that insect -derived factors are not required for this response. Current evidence t hus indicates that insect feeding generates long -distance electrical signals through the action of GRLs, and that decoding of this signal in systemic responding leaves results in JA -Ile synthesis, JA -Ile perception via the COI1 -JAZ co -receptor system and a ctivation of defense gene expression. The JATI -eliciting electrical signal thus has all the hallmarks of a DAMP (Figure 1 .2) The mechanism by which the propagating signal is perceived and subsequently linked to JA biosynthesis remains to be determined. How ever, there is evidence to suggest that calcium ions may be involved in propagating and/or interpreting the signal in responding target cells (Felle and Zimmermann 2007; Maffei et al. 2006; Qi et al. 2006) and a role for RBOHD -dependent ROS production was excluded (Mousavi et al. 2013). Negative Regulation of J ATI Although J ATI confers effective resistance to a broad spectrum of pathogens and herbivores , hyperactivation of the pathway can negatively affect plant gr owth and fitness (See Chapter 3 ). Many specialized defense compounds, for example, are toxic to the plant that produces them (Baldwin and Callahan 1993; Gog et al. 2005 ). In addition, increased allocation of limited metabolic resources to defense compounds may reduce the extent to which these resources can be used to fuel plant growth and reproduction (Agrawal 1999; Baldwin 1998; Herms and Mattson 1992; Yan et al. 2007; Zhang and Turner 2008) . J ATI may , therefore , provide a cost -saving strategy to coordinate the timing of chemical defense produc tion with perceived threat s 21!from the environment . Until recently, relatively little attention has been paid to understanding mechanisms that restrain defense signaling pathways or desensitize plant cells to the presence of eliciting signals . As described b elow, molecular studies have elucidated several JA-induced negative feedback loops within the core JA signaling module . Catabolism of JA-Ile Ð The dependence of J ATI on intracellular accumulation of JA -Ile suggests that turnover of the hormone could provide a mechanism to attenuate JA responses. Initial support for this hypothesis came from studies showing that various oxidized and conjugated derivatives of JA -Ile a ccumulate in wounded leaves (Glauser et al. 2008 ; Miersch et al. 2008; Paschold et al. 2008) . Recent studies have employed genetic approaches to elucidate two metabolic routes for JA -Ile catabolism, referred to here as the JA-Ile "-oxidation and hydrolysis pathways (Fig ure 1.2). The latter pathway is c atalyzed by aminohydrolases that cleave JA -Ile to JA and Ile (Bhosale et al. 2013; Widemann et al. 2013; Woldemariam et al. 2012; ). This reaction is readily revers ible by the JA -conjugating enzyme JAR1 (Staswick and Tiryaki 2004 ), suggesting that the relative level of conjugating and aminohydrolase activity is an important factor in the control of JA-Ile homeostasis. In contrast to JA -Ile hydrolysis, the "-oxidation pathway provides a mechanism for permanent inactivation of JA -Ile. This pathw ay involves at least two members of the CYP94 family of cytochromes P450 (CYP94B3 and CYP94C1) that oxidize the "-carbon of JA -Ile to produce 12OH -JA-Ile, which is further oxidized to 12COOH -JA-Ile (Heitz et al. 2012; Kitaoka et al. 2011; Koo et al. 2011 ). 12OH -JA-Ile is less active than JA-Ile in promoting COI1 bind ing to JAZ proteins (Koo et al. 2011) . The fact th at 12OH -JA-Ile retains some activity in COI1 -JAZ interaction assays, however, suggests that CYP94 -mediated 22!oxidation of 12OH -JA-Ile to 12COOH -JA-Ile, or conjugation of 12OH -JA-Ile to othe r small molecules (Gidda et al. 20 03; Kitaoka et al. 2014), is requir ed for complete inactivation of JA -Ile . Consistent with a role in negative feedback regulation of J ATI, genes encoding enzymes in both the "-oxidation and hydrolysis pa thways are rapidly induced in response to wounding, herbivory, and JA treatment (Bhosale et al. 2013; Heitz et a l. 2012; Kitaoka et al. 2011; Koo et al . 2011; Widemann et al. 2013 ; Woldemariam et al. 2012 ). Remarkably, Bhosale et al. (2013) found that the IIL6 gene encoding a JA -Ile aminohydrolase in Arabidopsis is co -expressed with other JA -response genes in pla nts grown under tightly controlled growth conditions in which stress treatment s were not intentionally imposed . This finding highlights the exquisite sensitivity of J A-associated surveillance and response systems, and suggests a broader role for JA signali ng in modulating phenotypic plasticity in response to subtle changes in the environment . It can be anticipated that future research will uncover mechanisms by which JA responses are integrated with various environmental perturbations, including changes in light, water status, nutrient availability, soil microbe communities and wind/touch ( e.g. Chehab et al. 2012). Stable JAZ proteins Ð A hallmark of most JAZ genes is rapid and strong expression in response to exogenous JA or stress -induced accumulat ion of endogenous JA (Chung et al. 2008; Chini et al. 2007; Thines et al. 2007; Yan et al. 2007 ). This pattern of expression sugges ts that de novo synthesis of JAZ proteins is part of a negative feedback system to desensitize cells to the presence of the hormone. Such a mechanism of feedback control, however, would depend on the existence of JAZ proteins that are relatively stable in presence of JA -Ile. Whereas initial studies demonstrated that some JAZ proteins (e.g., JAZ1) are rapidly degraded (t 1/2 < 2 min) in the presence of JA -Ile (Chini et al. 2007; Grunewald et al. 2009; Pauwels et al. 2010; Thines et al. 23!2007), recent studies have advanced the concept that other JAZs exhibit a wide range of stability, which could allow fine -tuning of TF activity in response to fluctuatin g JA -Ile levels (Chung and Howe 2009; Chung et al. 2009, 2010; Shyu et al. 2012). The conserved Jas motif of JAZ proteins contains a degradation signal (degron) that binds COI1 in a JA -Ile-dependent manner (Katsir et al. 2008 b; Melotto et al. 2008 ; Sheard et al. 2010; Yan et al. 2007 ). Point mutations within the degron disrupt JAZ -COI1 interaction without affect ing JAZ binding to TFs, thereby stabilizing and enhancing the activity of the repressor (Melotto et al. 2008; Withers et al. 2012) . Natural sequence variation within degron also affects JAZ stability and associated physiologi cal outputs of J ATI (Shyu et al. 2012). JAZ8, for example, contains a non -canonical degron that evidently does not interact with COI1 in the presence of JA-Ile. As a consequence, JAZ8 maintains the ability to interact with target TFs and repress transcription through recruitment of a co -repressor complex . Stress -induced e xpression of JAZ8 may thus provide a mechanism to desensitize cells to the presence of JA -Ile (Shyu et al. 2012) . JAZ repressors are also stabilized by alternative splicing (AS) events that remove or modify the Jas motif and its associated degron. AS of JAZ10 pre -mRNA produces several splice variants that differentially interact with COI1 in the presence of JA -Ile. These isoforms of JAZ10 exhibit a range of stability in JA -stimulated cells and, when overexpressed in planta , attenuate JA signal outputs to varying degrees (Chung and Howe 2009; Ch ung et al. 2010; Moreno et al. 2013). A direct role for JAZ10 AS in negative feedback control of J A signaling is supported by the JA -hypersensitive phenotype of jaz10 null mutants, as well as the ability of specific JAZ10 splice variants to complement the hyper sensitive phenotype of jaz10 mutants (Cerrudo et al. 2012; Demianski et al. 2012; Moreno et al. 2013; Yan et al. 2007 ) (See Chapter 2 ). The AS event responsible for generating the stable JAZ10.3 isoform involves retention of an intron whose 24!location within the Jas motif results in truncation of the C -terminal end of the motif. Interest ingly, this intron is present in most JAZ genes from phylogenetically diverse land plants , suggestin g that this conserved AS event provides a general mechanism to desensitize c ells to the presence of high JA-Ile levels (Chung et al. 2010). It remains to be determined how stable JAZ repressors are removed from cells in order to reset full sensitivity of the JA response. Transcriptional JAMming Ð A third mechanism to negatively regulate JA responses involves a phylogenetic clade of b HLH-type proteins (JAM1/bHLH017, JAM2/bHLH013 , JAM3/bHLH003) that is closely related to the positively acting MYC2 TF and its functional paralog s, MYC3, and MYC4 (Fonseca et al. 2014 ; Nakata et al. 2013; Sasaki -Sekimot o et al. 2013; Song et al. 2013 ). JAM proteins compete with MYC2 for binding to cis-acting G -box elements within the promoters of JA -responsive genes. However, because they lack the conserved activation domain found in MYC2/3/4, JAMs function as transcriptional repressors rather than activators. JAM TFs also interact directly with JAZ pr oteins, which may serve to increase the strength o f transcriptional repression th rough recruitment of the co -repressors NINJA and TOPLESS ( Fonseca et al. 2014; Song et al. 2013 ). Similar to other negativ e feedback loops , the expression of JAM1 is strongly upregulated by JA treatment and associated stress responses (Fonseca et al. 2014; Nakata et al. 2013; Sasaki -Sekimoto et al. 2013; Song et al. 2013). Other modes of negative regulation Ð The multiple negative feedback loops described above likely act in concert to restrain the amplitude and duration of JATI after the response is initiated. It should be noted, however, that the onset of JATI could be actively suppressed by other signals 25!when the benefit of growth outweighs the cost of defense . A compelling example is repression of JATI during the shade avoidance response in which changes in light quality , as perceived by the photoreceptor phytochrome B , modulates the stability of MYC TFs and JAZs to prioritize elongation growth over defense (Ballar” 2014; Cerrudo et al. 2012; Chico et al. 2 014; Moreno et al. 2009; Izzaguirre et al. 2013 ). Recent studies have also provide d insight into the mechanisms by which JATI is suppressed by the growth -related hormones gibberellic acid ( Hou et al. 2010; Yang et al. 2012 b) and ethylene ( Kim et al. 2014; Song et al. 2014) , as well as other transcriptional regulators whose mode of action is just beginning to be understood (Hu et al. 2013b). Manipulation of JATI by plant -associated organisms The efficacy of any given immune syst em is often reflected by the extent to which host -associated organisms evolved to evade that response . Consistent with its role in re-directing primary and secondary metab olism, perhaps it is not surprising that plant pathogens and herbivores evolved strategies to manipulate (acti vate or suppress ) JATI. Current views on this topic are influenced by the notion that JATI and SATI are often mutually antagonistic ( Caillaud et al. 2013; Kunkel and Brooks, 2002; Robert -Seilaniantz et al. 2011 ; Thaler et al. 2012 ). Studies performed with Arabidopsis , for example, have led to the generalization that increased activity of the JA sector of immunity enhances the virulence of biotrophic pathogens that are sensitive to SATI, whereas expression of SATI favors the performance of insect herbivores and necrotropic pathogens that are more sensitive to JATI. Compelling evidence for JATI -SATI antagonism comes from studies showing that many plant -associated organisms use effector -based strategies to create JA -SA imbalances that suppress JATI (Table 1.2). 26!An important emerging paradigm in plant -herbivore interactions is the ability of herbivore s to activate the SA pathway and thereby reduce the effectiveness of JATI as a basal defense (Hogenhout and Bos 2011; Walling, 2008). For example, p hloem feeding b y silverleaf whitefly ( Bemisia tabaci ) results in increased expression of SA-related defense genes and concomitant r epression of J ATI (Zarate et al. 2007 ; Zhang et al. 2013) . Similarly, insect egg -associated effector s trigger SA accumulation and JATI suppression i n host tissues surrounding the egg , thus favoring the survival of newly hatched larvae (Bruessow et al. 2010 ; Reymond 2013). Secretion of SA into the locomotion mucus (slime trail) by some molluskan herbivores (K−stner et al. 2014) , or excretion of SA into honeydew by some aphid species (Schwartzberg and Tumlinson 2013) , may reflect additional mechanisms to suppress JATI. The Coleopteran herbivore Leptinotarsa decemlineata (Colorado potato beetle) employs a n alternative but no less effective strategy to hijack J ATI (Chung et al. 2013) . Symbiotic bacteria in the oral secretion of the beetle activate SA-dependent responses and repress local and systemic JATI. That this phenomenon also occurs in a root -feeding insect herbivore ( Diabrotica virgifera, western corn rootworm) of maize suggest s that host defense suppression by symbiotic bacteria may be a general feeding strategy adopted by insect herbivores (Barr et al. 2010) . Studies of the Arabidopsis -Pseudomonas syringae strain DC3000 ( Pst DC3000) pathosystem have provide d considerable insight into how bacterial pathogen s manipulate JA-SA antagonism to their own advantage. In this system, immunity to Pst DC3000 is mediated in large part by SATI. Interestingly, Pst DC3000 uses multiple effectors to activate JA responses through targeted destruction of JAZ proteins , which in turn suppress es SATI (Fig ure 1.2). One well -studied effector is the polyketide coronatine (COR) that acts as a potent agonist of the COI1 -JAZ co-receptor system (Bender et al. 1993; Katsir et al . 2008 b; Sheard et al. 2010 ). COR -induced 27!degradation of JAZ repressors strongly upregulates the expression of JA -responsive defense genes and downregulates growth -related genes, and impairs multiple aspects of SATI (Attaran et al. 2014; Brooks et al. 2005; Melotto et al. 2006; Uppalapatti et al. 200 7; Zhao et al. 2003; Zheng et al. 2012 ;). Suppression of SATI by COR is mediated in part by NAC -type TFs that concomitantly repress the expression of the key SA biosynthetic enzyme ICS1 and activate expression of a methyltransferase (BSMT1) that converts SA to volatile MeSA (Attaran et al. 2009, 2014 ; Zheng et al. 201 2). That release of MeSA is also observed in other plant -enemy interactions (Dempsey et al., 2011) suggests that JA-induced disposal of SA through volatilization of MeSA may be a gene ral mechanism to antagoni ze SATI by stresses that trigger JA signaling . Pseudomonas syringae strains produce at least two type III secreted protein effectors that also promote degradation of JAZ proteins to increase pathogenicity . HopZ1a is a putative acetyltransferase that modifies JAZ proteins to stimulate their degradation in a COI1 -dependent manner (Jiang et al. 2013). HopX1 is a cysteine protease that destroys JAZs in dependently of COI1 (Gimenez-Ibanez et al. 2014) . Interestingly, HopX1 is produced by a strain of P. syringae that does not synthesize COR, indicating that distinct mechanisms to activate JA signaling through proteolytic destruction of JAZs have arisen independently in the evolution of this pathogen (Gimenez -Ibanez et al. 2014) . These findin gs are consistent with results of large -scale protein -protein interaction screens showing that JAZs are targets of effectors from both P. syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Mukhtar et al. 2011). In contrast to other biotrophic organisms , colonization of host tissues by some mutualistic ectomycorrhizal fungi is inhibited by JATI (Plett et al. 2014a). A recent study showed that the ectomycorrhizal fungus Laccaria bicolor produces an effector (MiSSP7) that binds to and 28!stabilizes a host JAZ protein to repress JA responses that p resumably inhibit establishment of the symbio sis (Plett et al. 2014b). These collective studies highlight the COI1 -JAZ co -receptor system as a central hub of plant immunity and portend the discovery of additional effectors from other plant -associated organisms that target the core JA module . Summary and future perspectives Recent research on many fronts has tremendously advanced our understanding of t he mechanism of JA signaling and its relationship to induced plant immunity . These efforts have coalesced around a simple model (Fig ure 1.2) to explain how fluctuating levels of a small-molecule hormone (JA -Ile) exert transcription al control over complex morphological and chemical defense traits . We suggest that the modular structure of JATI allows the conserved core JA module to link different combinations of PRR -based recognition systems (inputs) and defense traits (outputs) to create new specificities of host resistance . Indeed, there is a good evidence that JATI is a significant driving force in shaping plant -animal associations in natural environments (Kallenbach et al. 2012; Zt et al. 2012). This conceptual framework provides a foundation for studies aimed at understanding the underlying mec hanisms by which recognition -response systems give rise to phenotypic plasticity, and for revealing how interactions between the environment and the genome have spawned highly diverse, idiosyncratic defense traits in the plant kingdom. Meeting this challen ge will require integrative approaches sp anning the ecos ystems -to-gene continuum , as applied to experimental systems that offer both genomic and ecological resources. With the exception of a few model plants, remarkably little is known about the identity of JA-regulated compounds that provide resistance against specific attackers. It is currently unclear, 29!for example, whether JA/COI1 -mediated resistance of Arabidopsis, tomato, and maize to soil -borne Phythium spp (Table 1.1) involves similar or divergent suites of defense traits . M ajor differences in specialized defense chemistry between these species , however, suggests that different plants use a conserved core JA module to deploy different suites of chemical defense against the same broad host -range attacker. Similarly, there is evidence that tomato and Arabidopsis use distinct JA -regulated defense chemistry for protection agai nst the two -spotted Tetranychus urticae (spider mite ) and Tricho plusia ni (cabbage looper) (Herde and Howe 2014; Li et al. 2002 , 200 4; Z hurov et al. 2014). The modular architecture of JATI thus appears to support the evolution, in different host plants, of independent chemical solutions to the same pathogen or herbivore, which may contribute to the diversity and sporadic distribution of secondary metabolites in higher plants (Fraekel 1959). On the other hand, there are several examples of similar defense compounds that evolved independently in diverse plant families (Berenbaum and Zangerl 2008). Modern omics -based technologies offer tremendous potential to bee ter understand the evolution of constitutive and induced defense compounds by elucidating gene -pathway -metabolite relationships in diverse group of plants (Berenbaum and Zangerl 2008; Kliebenstein 2012). Insight into the evolutionary forces that drive the diversity of chemical defenses also will benefit from a better understanding of how these defense systems are matched by equally complex counter -defenses in plant attackers (Herde and Howe 2014). It is becoming increasingly evident that the JA/COI1/JAZ/TF module is a convergence point for direct cross talk with other signaling pathways that control growth and development (Ballar” 2014; Erb et al. 2012; Huot et al. 2014). It appears these crosstalk s occur primarily through direct interaction between nuclear factors that regulate transcription , including the Mediator c omplex ( Caillaud et al. 2013; Kidd et al. 2011 ). Future research aimed at 30!understanding changes in chromatin structure, epigenetic modificati on, and cis-regulatory codes (Zou et al. 2011) that direct TF -DNA interactions is expected to provide new insight into how transcriptional networks control complex JATI outputs, including transgenerational immunity (Rasmann et al., 2012) . Various negative feedback loops act in concert to restrain JATI outputs, but whether these control mechanisms constitute an adaptive response to balance tradeoffs between JATI and growth, or perhaps other forms of immunity, remains to be determined. Knowledge of how protei ns in different signaling pathways functionally interact to regulate growth -defense antagonism has potential practical application in the development of crop varieties that are both h igh yielding and stress tolerant (Shyu and Brutnell 2015) . These efforts may be aided by mathematical models to predict how environmental inputs are integrated within phytohormone network s to generate specific physiological outcomes (Middleton et al. 2012) . A significant gap in our understanding of JATI is how recognition of a danger signal at the cell surface activates JA biosynthesis. By analogy to stress -responsive regulation of ethylene biosynthesis (Liu and Zhang 2004) , identification of direct targets of the relevant MAPK cascades may provide important clues. Attention should also be given to the hypothesis that JA biosynthesis is controlled by calcium -dependent signaling event s that are coupled PRR activation (Romeis and Herde 2014) . Further analyses of how GLRs generate and/or propagate long -distance electrical signals will undoubtedly yield important new insight s as well. A systems -level understanding of JA-Ile homeostasis , including pathways by which JAs are transported within and between cells , is ultimately needed to understand how specific TFs are controlled by thresholds and time -dependent signatures of the hormone. Finally, it should be noted that although there is molecular evidence that plant resistance to insect herbivore is mediated by 31!PRRs (Abuqamar et al. 2008; Prince et al. 2014; Truitt et al. 2004; Yang et al. 2011), HAMP receptors remain to be identified in any plant. One of the most exciting recent advances in the field of induced immunity is evidence that the core JA module is a common target of effectors from multiple plant -associated microbes (Table 1.2). This finding is consistent with the idea that different pathogens independently evolved virulence effectors that converge on common host targets within the PTI network (Jones and Dangl 2006; Mukhtar et al. 2011). Only time will tell w hether the current list of COI1/JAZ -targeting effectors is complete or, more likely, will continue to expand as effector repertoires from diverse plant -associated microbes , insects , and nematodes are systematically scrutinized (Boller and He 2009; Elzinga and Jander 2013; Hogenhout and Bos 2011; Kandoth and Mitchum 2013). The s trong selection pressure imposed by JATI on arthropod herbivores and necrotrophic pathogens, together with evidence that these organisms actively suppress JA -based defenses , suggests the existence of novel mechanisms by which plant - associated organisms disrupt JATI . Interdisciplinary approaches aimed at understanding how the JA module promotes broad -spectrum immunity through the control of specialized metabolism, and how this branch o f immunity is subverted by plant attackers, offer tremendous potential to help solve pressing problems facing the world (Plant Science Research Summit 2013). From a biotechnological perspective, for example, these efforts may inform synthetic approaches to harness specialized biochemical pathways for metabolic engineering of new chemistries for a variety of plant -based products, including pigments, fragrances, flavors, pesticides and pharmaceuticals. Given the current pace of discovery and technological too ls available, exciting new discoveries may be just around the corner. 32! Acknowledgements We thank Marlene Cameron for assistance with figure graphics and the MSU Diagnostic Lab for diagnosis of Pythium spp -media ted root rot disease on jai1 tomato plants. This work was supported in part by the National Institu tes of Health (grant no. GM57795), the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy (grant no. DE ÐFG02 Ð91ER20021) , and a College of Natural Science Dissertation Continuation Fellowship to M.C. 33! REFERENCES 34! 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Patel. 57!Abstract Jasmonates (JAs) are essential orchest rators of plant development, controlling a myriad of growth and defense processes. A fundamental step in the a ctivation of the JA pathway involves the JA -dependent degradation of JASMONATE ZIM -domain (JAZ) transcriptional repressors through the activity of the E 3 ubiquitin -ligase SCF COI1 . It is now becoming evident that events of alternative splicing in the JAZ re pressors expand the repertoire of regulatory proteins modulating JA responses . Alternative splicing in the Arabidopsis thaliana JAZ10 gene generates three protein isoforms with remarkable differences in their degree of stability upon JA -mediated degradatio n, but whose biological relevance is still poorly u nderstood. We here show that two stable splice variants of JAZ10 (JAZ10.3 and JAZ10.4 ) are involved in attenuation of JA signaling, ac ting as dominant repressors to regulate the amplitude and duration of the JA responses. Complementation experiments performed with the jaz10-1 mutant indicate that, u pon induction, JAZ10.3 and JAZ10.4 are retained in plant cells for longer periods of time to downregulate JA -related pro cesses such as the expression of defense -related genes and the JA -mediated inhibition of root elongation. T his JA -desensitization is mainly performed by JAZ10.3, the most abundant protein produced from JAZ10 . Structural analysis showed that JAZ10.3 stabili ty is caused by a single amino acid disruption in its degron sequence . The observation that JAZ10.3 -like genes are found in phylogenetically diverse plant species highlights that these stable repressors are essential for proper regulation of the JA pathway . Moreover, the observation that JAZ10 functions are not shared with other JA -stable JAZ genes highlights a degree of functional specificity among the JAZ family members. In conclusion, o ur results illustrate how plants utilize specific sets of JAZ repress ors to precisely regulate the JA signaling pathway and optimize plant fitness under different environmental conditions. 58!Introduction Jasmonates (JAs) are a class of fatty -acid derived hormones controlling diverse aspects of plant physiology. Besides its widely studied ability to promote plant defense against a plethora of enviro nmental stresses (Campos et al. 2014; Howe and Jander 2008; Wasternack and Hause 2013), JAs also play a role in growth processes such as cell differentiation and division, seed germination, root and shoot growth, flower formation, senescence, a mong many others (Browse et al. 2009; Wasternack et al. 2013; Wasternac k and Hause 2013). The idea that these lipid -derived molecules evolved as essential modulators of developmental plasticity is supported by its ubiquitously occurrence throughout the plant kingdom and its extensive crosstalk with other plants hormones to me diate virtually every aspect of plant bi ology (Ballar” 2011; Erb et al. 2012, Hamberg and Gardner 1992; Song et al. 2014; Yang e t al. 2012). Thus, it is not surprising that elaborate genetic networks have evolved to regulate the JA responses in order to op timize plant fitness under different environmental conditions. A major regulatory step in activation of JA responses involves the removal of the JASMONATE ZIM -domain (JAZ) proteins, transcriptional repressors that negatively regulate the hormone response s by binding to and inhibiting transcription factors such as MYC2 (Chini et al. 2007; Fern⁄ndez -Calvo et a l. 2011; Thines et al. 2007; Yan et al. 2007). Upon elicitation by environmental signals, a burst in the endogenous levels of the bioactive form of th e JA, jasmonoyl -isoleucine (JA -Ile) , promotes the association of the JAZ proteins with the CORONATINE INSENSITIVE1 (COI1) component of the SCF COI1 ubiquitin E3 ligase complex, leading to ubiquitination and degradation of the JAZ by the 26S proteasome, and further release of transcription factors (TFs) from repression to activate t he JA responses (Chung and Howe 2009; Melotto et al. 2008; Xie et al. 1998). Biochemical and structural studies demonstrate that 59!the formation of the CO I1-JAZ complex is dependent on the conserved Jas motif, located at the C-terminal end of the JAZ proteins ( Katsir et al. 2008; Melotto et al. 2008; Sheard et al. 2010). A short 21 amino -acid sequence within this motif defines the ÒdegronÓ, the minimal peptide necessary and sufficient for COI1 -JAZ physical interaction. The degron peptide adopts a bipartite loop/ #-helix structure that physically traps the JA -Ile molecule inside the COI1 ligand -binding pocket to form the JA co -receptor complex (Sheard et al. 2010). Accordingly , artificia lly truncated JAZ variants lacking the degron sequence are unable to interact with COI1 in a JA -Ile dependent manner, therefore being resistant to degradation through th e 26S proteasome ( Chini et al. 2007; Thines et al. 2007; Yan et al. 2007). Plants ectop ically expressing these JA -stable JAZ repressors exhibit decreased sensitivity to exogenous JA, increased susceptibility to insect feeding, improper development of reproductive organs and reduced production o f secondary metabolites (Browse 2009; Chung et al. 2008; Thines et al. 2007; Yamada et al. 2012; Yan et al. 2007). The finding that si milar modifications in the JAZ d egron sequence also occur naturally indicate the existence of a complex layer of regulatory mechanisms modulating JA responsiveness, whose role and contribution to plant phenotypic plasticity are still poorly understood. It was recently demonstrated that a conserved clade of JAZ proteins lacks the canonical degron sequence and weakly associate with COI1 in the presence of JA -Ile (Shyu et al. 2012; Thireault et al. 2015 ). Due to their stability against JA -mediated degradation, JAZ8 and JAZ13 are more stable in JA -elicited cells and are capable of repressing JA responses in the presence of high concentrations of the hormone. The occurrence of these inherently stable JAZ s in phylogenetically distinct plant species implies that these degron -altered JA -stable rep ressors 60!are fundamental for proper function of the JA pathway (Pirrello et al. 2014; Shyu et al. 2012; Thireault et al. 2015; Wang et al. 2014). Alternative splicing is a fundamental process underlying the increased cellular and functional complexity in eukaryotes. As a molecular mechanism to ge nerate multiple mature mRNAs from a single gene, alternative splicing is the main mechanism expanding proteom e diversity in complex genomes (Ben -Dov et al. 2008; Graveley 2001). Recent analysis in numerous species indicates that alternative splicing in plants is a more prevalent process tha n previously thought (Li et al. 2014; Mandadi and Scholthof 2015; Marquez et al. 2012; Shen et al. 2014; Thatcher et al. 2014). In Arabidopsis thaliana , for example, 60% of the in tron -containing genes are alternat ively spliced (Filichkin et al. 2010; Marquez et al. 2012). The application of high -throughput sequencing and studies using different organs, developmental stages and external conditions continues to reveal new splice vari ants in plant genomes (Mandadi and Scholthof 2015; Syed et al. 2012). However, examples demonstrating the function and biological relevance of different splice isoforms are still scarce. Analysis of phylogenetically diverse species indicates that alternati ve splicing is a common feature of th e JAZ gene family (Chung et al. 2010; Pirrello et al. 2014). The importance of this process as a post -transcriptional mechanism to control JA responses is evidenced by the recurrence of splicing events that modify the J as domain and affect JA -mediated C OI1 interaction (Chung and Howe 2009; Chung et al. 2010; Moreno et al. 2013; Pirrell o et al. 2014 ; Yan et al. 2007 ). The structure of numerous JAZ genes in different plant species includes a conserved Jas intron whose retention through alternative splicing generates proteins with a truncated Jas domain ; these splice isoforms interact weakly with COI1 in the p resence of JA -Ile ( Chung and Howe 2009; Chung et al. 2010). Other alternative splicing events create JAZ isoforms that lack the entire JAZ degron and thus are 61!unable to interact with COI1 in the presence of the hormone (Chung and Howe 2009; Moreno et al. 2013; Yan et al. 2007). Together, these events generate a repertoire of JAZ variants that can possibl y respond differently to dyna mic range of JA concentrations. This hypothesis remains to be tested and u ncovering the role of this repertoire of repressors may improve our understanding of the regulatory mechanisms controlling the JA signaling pathway and h ow hormone responses are finely tuned in response to changing environmental conditions . At least nine of the 13 JAZ genes in Arabidopsis are subject to alternative splici ng (Chung et al. 2010) . JAZ10 is the best example of how this post -transcriptional me chanism increases the functional diversity in this family. The JAZ10 pre -mRNA is spliced to produce three protein isoforms that differ in their stability due to the varying length of their Jas domain (Figure 2.1A): JAZ10.1 carries a full -length Jas domain , strongly interacts with COI1 and , as a consequence, is rapidly degraded in the presence of low concentra tions of JA -Ile (Chung and Howe 2009; Shyu et al. 2012). JAZ10.3 is generated through an intron retention event that results in a partial truncation of the Jas domain; this isoform weakly interacts with COI1 and is stable in JA -elicited plant cells (Chung and Howe 2009; Yan et al. 2007). Finally, utilization of an alternative splic e donor in third exon of JAZ10 leads to a frame -shift mutation that removes the entire Jas domain, creating a highly stable isoform (JAZ10.4) that does not int eract with COI1 (Chung and Howe 2009; Moreno et al. 2013; Yan et al. 2007). Although the biochemical features of JAZ1 0.3 and JAZ10.4 suggest a role for these protein s in attenuation of JA responses in sensitized cells (Chung and Howe 2009; Moreno et al. 2013), the biological relevance of JAZ10 alternative splicing remains largely unknown . Here we describe the wound - and JA -induced accumulation of alternatively splic ed JAZ10 transcripts and their corresponding protein isoforms. We show that, al though JAZ10.1 is 62! Figure 2.1. The Arabidopsis thaliana JAZ10 gene is subjected to alternative splicing. (A) Schematic diagram of alternative spliced JAZ10 transcripts and their corresponding protein isoforms. White and black bars in the gene models represent UTRs and coding sequences, respectively. Splice variant -specific primers used for qRT -PCR experiment are denoted as arrows below gene models. Dotted lines indicate prim ers that span a splice junction. The cryptic MYC2 -interacting domain (CMID), ZIM and Jas domains are represented, respectively, by green, yellow and blue boxes in the protein structure. (B) Expression of JAZ10 splice variants in response to mechanical woun ding. Wounded leaves were collected at various time points after wounding. Data was normalized to JAZ10.1 levels at the 0 h time point (relative expression). As controls, leaves were harvested immediately before the start of the experiment (Ò0Ó). Bars deno te mean ± S.D. of three biological replicates with three technical replicates each. (C) Ratio of JAZ10.2 , JAZ10.3 and JAZ10.4 over JAZ10.1 showed by green, red and blue bars, respectively. Values were obtained using the expression data shown in (B). 63! the most abundant transcript produced from splicing of the JAZ10 pre -mRNA, JAZ10.3 is the most abundant protein isoform to accumulate in response to elicitation. We also describe a genetic complementation system to assess the relative contribution of each JAZ alternatively splicing variant in various JA -mediated physiological responses, and we us e this assay to demonstrate that other stable JAZ repressor s are not functionally equivalent to JAZ10. Finally, we provide insight into the molecular mechanism by whic h the abundant JAZ10.3 isoform represses JA signaling, and propose that this mechanism of alternative splicing -induced JAZ stabilization is generally conserved throughout the plant kingdom. Results Expression dynamics of JAZ10 splice variants in response to wounding As is the case for most JAZ genes in Arabidopsis, JAZ10 is transcriptionally activated in response to tissue damage and other stimuli that trigger the biosynthesis of JA -Ile (Chung et al. 2008; Demianski et al. 2012; Mousavi et al. 2013; Yan et al. 2007). We used quantitative Real -Time PCR (qRT -PCR) to determine the expression pattern and relative abundance of each of the alternatively spliced JAZ10 transcripts in local (damaged) and systemic (undamaged) leaves of wounded plants (Figures 2.1 and 2.2). W ounding induces a rapid but transient increase in all four JAZ10 transcripts in locally damaged leaves. The level of each transcript peaked one hour after wounding and, at this point, was at least 100 -fold greater than that in unwounded leaves (Fig ure 2.1B). JAZ10.1 was consistently the most abundant (~50% of total JAZ10-derived mRNA) of the alternatively spliced transcript at each time point analyzed. The level and time -dependent pattern of accumulation of JAZ10.2 and JAZ10.3 were remarkably simil ar to each other, and together accounted for 40 -45% of total JAZ10 transcripts. JAZ10.4 was the least abundant transcript, 64! Figure 2.2. Schematic representation of the wounding experiment. (A) Four week -old Arabidopsis plants were mechanically wounded with a hemostat. Wounded leaves (red) were selected based on their position in the stem. Non -wounded (systemic) leaves (green) were also harvested. (B) Timeline for the wounding experiment. Time of day and time when tissue was collected (and wounding performed) are depicted. White and black bars in the timeline indicate light and dark periods, respectively. Three plants were pooled for each time point an d the experiment was repeated three independent times with similar results. 65! accounting for only 5 to 10% of total JAZ10 mRNA. Consistent with the general pattern of expression shown in Figure 2. 1B, the relative proportion of the four JAZ10 transcripts r emained constant du ring the time course (Figure 2.1 C), suggesting that the alternative splicing of JAZ10 pre-mRNA is likely a non -regulated process. The temporal dynamics and relative abundance of JAZ10 transcripts in undamaged leaves of wounded plants (systemic response) was remarkably similar to the local response, with the exception that the absolute level of JAZ10 mRNA is systemic leaves was ~10 -fold less than that in wounded leaves (Figure 2.3) To f urther test the hypothesis that alternative splicing of JAZ10 pre-mRNA is a non -regulated process, we quantified the relative expression level of splice variants in flowers and roots of a transgenic line ( 35S:JAZ10G ) that expresses a genomic copy of JAZ10 from the constitutive 35S promoter of Cauliflower Mosaic Virus (CaMV) (Chung et al., 2010). In good agreement with the analysis of wounded leaves, the results showed that in both roots and flowers, JAZ10.1 and JAZ10.4 were the most and least abundant, resp ectively, JAZ10 tran script , whereas the combined level of JAZ10.2 and JAZ10.3 was comparable to JAZ10.1 (Figure 2.4). Collective ly, these results indicate that, regardless of the level of pre -mRNA expression, tissue type and mode of induction, alternative splicing of JAZ10 pre -mRNA results in the production of four splice variants whose relative proportion remain constant. Dynamics of JAZ10 protein splice variant accumulation in response to wounding JAZ10 splice variants differ from each other in the lengt h of the Jas domain, which harbors the COI1 -interacting degron sequence that mediates JA -dependent degradation of JAZ proteins (Chung and Howe 2009; Moreno et al. 2013). A second major factor influencing JAZ10 protein accumulation is JA -dependent transcrip tional activation of the JAZ10 gene . Moreover, since 66! Figure 2.3. Wounding systemic expression of JAZ10 transcripts. (A) Unwounded (systemic) leaves were harvested for RNA extraction at the indicated times after wounding. Transcript levels were quantified by qRT -PCR as described above. Bars denote mean ± S.D. of two biological replicates with three technical replicates each. (B) Ratio of JAZ10.2 , JAZ10.3 and JAZ10.4 over JAZ10.1 showed by green, red and blue bars, respectively. Values were obtai ned using the expression data shown in (A). 67! Figure 2.4. Quantification of JAZ10 transcripts in flowers and roots of 35S:JAZ10G transgenic plants. (A) Relative expression of JAZ10 splice variants in flowers and roots of transgenic plants overexpressing the JAZ10 genomic sequence from the 35S promoter ( 35S:JAZ10G ). Data was normalized to reference genes and bars denote mean ± S.D. of three biological replicates with three technical r eplicates each. (B) Ratio of JAZ10.2, JAZ10.3 and JAZ10.4 over JAZ10.1 in different tissues is showed by green, red and blue bars, respectively. Values were obtained using the expression data shown in (A). 68! alternative splicing can lead to selective deg rada tion of transcripts through non sense -mediat ed mRNA decay (Wang and Brendel 2006), it is unclear whether all alternatively spliced JAZ10 transcripts are indeed translated . To clarify these questions , we fused a 2.0 kb JAZ10 promoter fragment ( JAZ10p ), which is sufficient to confer JA -inducibility to a reporter gene (Moreno et al. 2013; Sehr et al. 2010) , to the genomic sequence of JAZ10 (JAZ10g Ð Figure 2.5A ). Placement of a hemagglutinin (HA) epitope tag at the N -terminus of the protein allowed detec tion of all JAZ10 protein splice isoforms that share same N -termini but differ in the length of the C -terminus (Figure 2.5 B). The resulting JAZ10p:HA -JAZ10g transgene was transform ed into the jaz10 -1 mutant (Demianski et al. 2012) for production of stable transgenic lines . Immunoblot experiment s performed with protein extracts from wounded leaves of such line showed that that all three JAZ10 protein isoforms accumulate within 1h of mechanical wounding , with pr otein levels peaking at the 2 h time point (Figure 2 .6A). Although JAZ10.1 was the most abundant transcript in wounded leaves (Figure 2.1B), we found that the corresponding JAZ10.1 protein was significantly less abundant than JAZ10.3 throughout the time course. At the 16 -h time point, for example, the JA -stable isoforms JAZ10.3 and JAZ10.4 were both detected whereas JAZ10.1 was not. All three variants were undetectable at the 24 h time point, indicating the existence of mechanisms to efficiently remove even those isoforms (e.g. JAZ10.3) that are st able in the presence of JA. To further assess the expression dynamics of JAZ10 splice variants in wounded leaves, we also expressed individual HA -tagged JAZ10 cDNAs ( JAZ10.1, JAZ10.3 or JAZ10.4) from the native JAZ10 promoter in the jaz10-1 mutant backgro und (Figure 2.5A ). Wounding experiments performed with the resulting in JAZ10p:HA -JAZ10.1 , JAZ10p:HA -JAZ10.3 and JAZ10p:HA -JAZ10.4 lines showed a similar trend as observed in the JAZ10p: HA-JAZ10g . For example, 69! Figure 2.5. Constructs used to study protein dynamics of JAZ10 splice variants in planta . (A) Schematic representation of the constructs used to study protein dynamics. A 2 kb region comprising the JAZ10 promoter was fused to the genomic sequence of JAZ10 (JAZ10g ) o r the cDNA of JAZ10.1 , JAZ10.3 and JAZ10.4 . The hemagglutinin (HA) -tag sequence used for immunoblot detection is shown in red. Constructs were used to transform the jaz10-1 mutant (see Methods). (B) Tricine -SDS-PAGE gels (Sch−gger, 2006) were used to resolve JAZ10 protein splice variants. Samples were obtained from leaf tissue of jaz10-1 plants transformed with constructs in (A), one hour after mechanical wounding. JAZ10 splice variants were detected with an #-HA antibody. 70! Figure 2.6. Dynamics of JAZ10 splice variant accumulation in response to mechanical wounding. (A) Accumulation of JAZ10 protein variants in response to mechanical wounding. Rosette leaves of JAZ10p:HA-JAZ10g in the jaz10-1 background were mechanically wounded with a hemostat. Wounded leaves were harvested for protein extraction at the indicated time after wounding (TAW). As a control, leaves were harvested immediately prior to wounding (0). A Coomassie blue -stained membrane (CB ) is shown as a loading control. (B) Wound induced accumulation of JAZ10 protein in lines expressing individual splice isoforms ( JAZ10p :HA-JAZ10.1 , JAZ10p :HA-JAZ10.3 and JAZ10p :HA-JAZ10.4 ). A Coomassie blue -stained membrane (CB) is shown as a loading contr ol. TAW, Time after wounding; NS, Non -specific band. 71! JAZ10.1 peaked at 2 h post wounding and then declined to undetectable levels at the 8 h time point (Figure 2 .6B). Conversely , the JA -stabl e variants JAZ10.3 and JAZ10.4 we re also rapidly induced, but were retained for the duration (16 h) of the time course. The high level of wound -induced JAZ10.4 accumulation in JAZ10p:HA -JAZ10.4 relative to that in the JAZ10p:HA -JAZ10g lines likely reflects the absence, in the formed line, o f alternative splicing control mechanisms that limit the production of JAZ10.4 -encoding mRNAs, as observed for the endogenous JAZ10 gene (Figure 2.6A) (Moreno et al. 2013). Taken together these results indicate that, upon induction by mechanical wounding, all three protein s splice variants of JAZ10 are rapidly (<1h) and transiently produced. However, differences in protein stability dictated by the COI -interacting Jas motif differentially affect the stability of each isoform, thus leading to the accumulati on of the more stable isoforms. The combined effect of transcript abundance and protein stability make JAZ10.3 the most abundant of the JAZ10 splice variant protein in wounded leaves. Stable isoforms of JAZ10 complement the JA-hypersensitive phenotype of jaz10 -1 Overstimulation of the JA signaling pathway is associ ated with fitness costs, including inhibition of growth processes ( Baldwin 1998; Yan et al. 2007; Yang et al. 2012; Zhang and Turner 2008). Thus, it is reasonable to hypothesize that mechanisms may exist to desensitize cells to JA or to and restrain the duration and amplitude of JA responses. The JA -hypersensitive phenotype of the jaz10-1 mutant further suggests that one or more JAZ10 splice variants may be involved in attenuation of JA responses . To test this hypothesis we first compared the root growth phenotype of the above -described JAZ10p:HA -JAZ10g to jaz10 -1 (Demianski et al. 2012). When grown for eight days on Murashige and Skoog (MS) medium supplemented with 72!20 µM of methyl -JA (MeJA), the root length of independent JAZ10p :HA-JAZ10g transgenic lines was significantly longer than that of jaz10 -1 seedlings but comparable to WT seedlings, indicating that JAZ10p:HA -JAZ10g complements the JA -hypersensitivity of jaz10 -1 roots (Figure 2.7A). To evaluate which splice variant is responsible for this effect , we next tested the ability of each individual JAZ10 splice variant to complement the JA -hypersensitive root growth phenotype of jaz10 -1. We found that the JAZ10p:HA -JAZ10.3 and JAZ10p:HA -JAZ10.4 tra nsgenes but not JAZ10p :HA-JAZ10.1 , reduce the sensitivity of jaz10 -1 roots to exogenous MeJA (Figure 2.7A). This finding supports the hypothesis that JA -induced expression of the stable JAZ10.3 and JAZ10.4 isoforms play a role in attenuation of JA responses, and further indicate that the JA -hypersensitive phenotype of jaz10 -1 is caused by the elimination of these two splice variants. We used immunoblot analysis to determine whether the ability of JAZ10 splice variants to attenuate JA -induced roo t growth inhibition correlates with the accumulation of the splice variants in JA -elicited plants (Figure 2.7B) . For this purpose, total protein extracted from eight day -old seedlings grown either in the presence or absence of MeJA was subjected to western blot analysis with an anti -HA antibody. The results showed that seedlings grown continuously in the presence of MeJA accumulate the stable JAZ10.3 and JAZ10.4 protein variants but do not accumulate detectable levels of JA -labile isoform JAZ10.1 . The absen ce of JAZ10.1 signal in the immunoblots is likely a consequence of the strength of interaction of this variant with COI1 and the prolonged exposure of seedlings (8 d) to MeJA treatment. Together, t hese results establish a causal relationship between the ac cumulation of specific JAZ10 splice variants and the attenuation of JA responsiveness. 73! Figure 2.7. Stable splice variants of JAZ10 complement the JA-hypersensitive root growth phenotype of jaz10 -1. (A) MeJA -induced root growth inhibition assay of wild type (WT), jaz10 -1, and jaz10 -1 lines transformed with the indicated transgenes. Root length was measured in eight -day old seedlings grown in MS medium supplemented or not with 20µM MeJA. The root length r atio was calculated by dividing the average root length of seedlings grown on medium containing MeJA by the average root length of the same genotype grown in MS medium lacking MeJA. Data show the mean ± S.E. of at least ten seedlings for WT and jaz10 -1 and >40 seedlings per transgenic line. Letters denote a statistical difference in comparison to WT control (TukeyÕs HSD, p-value <0.05). A minimum of seven independent lines were used to obtain the average shown for each transgenic line. (B) Accumulation of J AZ10 protein isoforms in seedlings of WT, jaz10-1 and jaz10-1 transgenic lines eight days after grown on the MS medium lacking ( -) or containing (+) of 20µM MeJA. Coomassie blue (CB) stained membrane is shown as a loading control. NS, non -specific band. 74! We used qRT -PCR to investigate the role of JAZ10 splice variants in attenuating the wound -induced expression of primary JA -response genes LIPOXYGENASE3 (LOX3) and 12-OXO-PHYTODIENOIC ACID REDUCTASE3 (OPR3) in leaves of adult plants (Chung et al. 2008). Ini tial comparisons between WT and jaz10 -1 plants showed that the latter genotype hyper -accumulates LOX3 and OPR3 transcripts in wounded leaves (Figure 2.8). This finding is consistent with previous studies of a jaz10 mutant generated by RNAi silencing (Yan e t al. 2007). Next we analyzed LOX3 and OPR3 expression in the same transgenic lines used for the root growth assays. The results showed that JAZ10p:HA -JAZ10g and JAZ10p:HA -JAZ10.3 restore wound -induced marker gene expression to levels seen in WT plants. Wo und-induced expression of both LOX3 and OPR3 in JAZ10p:HA -JAZ10.4 plants was significantly lower than in WT, which may reflect the high levels of wound -induced JAZ10.4 protein accumulation in these lines (Figure 2.6B). On the other hand, no complementation was observed in the transgenic (JAZ10p :HA-JAZ10.1 ) expressing the labile JAZ10.1 protein isoform (Figure 2.8). The se findings support the hypothesis that the up -regulation in LOX3 and OPR3 transcript levels in jaz10 -1 is caused by the absence of the stabl e JAZ10 repressors. To further test the idea that wound -induced production of stabilized variants of JAZ10 is sufficient to attenuate JA responses , we mutated a key Arg residue (R171 in JAZ10.1) within the degron sequence that mediates ligand -dependent interaction of JAZs to COI1 (Melotto et al. 2008; Sheard et al. 2010; Withers et al. 2012) . Yeast two -hybrid (Y2H) assays evidence that the R171A mutation disrupted JAZ10.1 -COI1 interaction, with the two proteins failing to interact even in the presence of high concentrations of the JA -Ile analog coronatine (Figure 2.9 ). To determine whether this stabilized form of JAZ10.1 can complement the JA -hypersensitive phenotype of jaz10 -1, we performed root growth inhibition assay with a jaz10 -1 line 75! Figure 2.8 . Stable splice variants of JAZ10 complement a transcriptional phenotype of jaz10 -1. Transcript levels of two biosynthetic genes ( LOX3 and OPR3) quantified by qRT -PCR using RNA extracted from leaves before ( -) and two hours after (+) mechanical wounding. Data represents expression relative to WT unwounded control. Bars denote mean ± S.E. of three biological replicates with three technical replicates each. Letters denote a statistical difference according to TukeyÕs HSD ( p-value<0.05). 76! Figure 2.9. R171A mutation disrupts JAZ10.1 -COI1 interaction. Yeast two -hybrid analysis of coronatine (COR) -mediated JAZ10.1 -COI1 interaction. Yeast strains were co -transformed with JAZ10.1 or JAZ10.1 R171A and COI1 and plated on medium containing different concentrations of COR. As negative and positive controls for protein interaction, yeast cells were also transformed with empty vector (EV) and JAZ10.1 respectively. 77! (JAZ10p:HA -JAZ10.1 R171A ) that expresses HA -JAZ10.1 R171A under the control of the JAZ10 promoter. Amon g eight independent JAZ10p:HA -JAZ10.1 R171A lines tested (T2 generation), all exhibited significantly reduc ed sensitivity to JA (Figure 2.10A ). Western blot analysis of protein extracts from two representative lines (H2 and H4) showed that the JA insensitivity is associated with induced accumulation of JAZ10.1 R171A . In contrast, and consistent with the results showed in Figure 2.7B, the labile WT form JAZ10.1 was not detected in JAZ10p:HA -JAZ10.1 grown continuously either in the presence or absence of MeJA (Figure 2.10 B). These results demonstrate that JA -induced expression of stable JAZ10 protein variants is sufficient to dampen the plantÕs sensitivity to JA. JAZ8 does not functionally complement stable isoforms of JAZ10 The Arabidopsis JAZ8 protein has also been described as a stable JAZ. Unlike stable isoforms of JAZ10, however, the stability of JAZ8 results from a non -canonical degron loop region that fails to interact with COI1 in the presence of JA -Ile (Shyu et al. 2012) . The domain archi tecture of JAZ8 and JAZ10 is also distinct with respect to the mechanism by which the two proteins recruit the co -repressor TOPLESS (TPL). The biological significance of these differences, however, remains unknown. We therefore designed an experiment to te st whether JAZ8 and JAZ10 are functionally equivalent. Specifically, we transformed the jaz10 -1 mutant with a HA -tagged derivative of JAZ8 expressed under the control of the JAZ10 promoter, and then tested the resulting JAZ10p:HA -JAZ8 lines for JA -induced root growth inhibition. Among seven independent transgenic lines (T2 generation) analyzed, none complemented the JA -hypersensitive phenotype of jaz10 -1 (Figure 2.11A). Western blot analysis performed with seedlings from representative JAZ10p:HA -JAZ8 lines (K13 and K15) showed that, despite the 78! Figure 2.10. A stabilized form of JAZ10.1 complements the JA -hypersensitive phenotype of jaz10 -1. (A) Root elongation assay on WT, jaz10 -1 and jaz10 -1 lines transformed with JAZ10p:HA -JAZ10.1 or its mutant form, R171A. Eight independent JAZ10p:HA -JAZ10.1 R171A lines (T2 generation) were used for the experiment. Root length was measured in eight -day old seedlings grown in MS medium supplemented or not with 20 µM MeJA. The root length ratio was calculated by dividing the average root length of seedlings grown on medium containing MeJA by the average root length of the same genotype grown in MS medium lacking MeJA. Data show the mean ± S.E. of at least ten seedlings for each genotype. Letters deno te a statistical difference in comparison to WT control (TukeyÕs HSD, p-value<0.05). (B) Accumulation of JAZ10.1 protein in JAZ10p:HA -JAZ10.1 R171A seedlings grown form eight days after on MS medium in the absence ( -) or presence (+) of 20 µM MeJA. Two ind ependent R171A lines (H2 and H4) were tested. Coomassie blue -stained membrane (CB) is shown as a loading control. 79! Figure 2.11. JAZ8 does not complement the JA -hypersensitive root growth phenotype of jaz10 -1. (A) Root growth inhibition with WT, jaz10 -1 and eight independent jaz10 -1 lines transformed with JAZ10p:HA -JAZ8. Root length was measured in eight -day old seedlings grown in MS medium supplemented or not with 20 µM MeJA. The root length ratio was calculated by dividing the average root lengt h of seedlings grown on medium containing MeJA by the average root length of the same genotype grown in MS medium lacking MeJA. Data show the mean ± S.E. of at least ten seedlings for each genotype. Letters denote a statistical difference in comparison to WT control (TukeyÕs HSD, p-value<0.05). (B) Accumulation of JAZ8 protein in eight day -old seedlings of jaz10 -1 and two jaz10 -1 transgenic lines (K13 and K15) carrying JAZ10p:HA -JAZ8 grown on the MS media in the absence ( -) or presence (+) of 20 µM MeJA. Co omassie blue -stained membrane (CB) is shown as a loading control. 80! absence of complementation, JAZ8 protein accumulated in a JA -dependent manner (Figure 2.11B). We conclude that JAZ8 does not functionally complement stable isoforms of JAZ10. Increased stability of JAZ10.3 is caused by alternative splicing -induced truncation of the Jas motif helix Given our result showing that JAZ10.3 plays a central role in attenuating JA responses, we next turned our attention to the mechanisms by which the intron ret ention event responsible for production of JAZ10.3, which is truncated at R185, weakens the interaction of this splice variant with COI1 (Figure 2.12A). Structural studies have revealed that the Jas motif harbors a bipartite JAZ degron sequence consisting of an N -terminal hormone -binding loop followed by an amphipatic #-helix that docks the JAZ proteins on the surface of COI1 (Sheard et al. 2010). Because JAZ10.3 contains an intact hormone -binding loop (Figure 2.12A), we hypothesized that the weak COI1 -JAZ10.3 interaction results from modification of the C -terminus of the Jas motif #-helix. In structural studies of the JAZ1 degron, Sheard et al. (2010) noted that hydrophobic residues near the N -terminal end of the Jas motif helix cluster on one face of the h elix to form a hydrophobic interface with COI1. The potential contribution of the C -terminal end of the helix, however, was not resolved in the x -ray crystal structure. Helical wheel plots of the JAZ1 and JAZ10 Jas motif helix reveals that V220 and L179 in JAZ1 and JAZ10, respectively, are embedded together with two highly conserved Leu residues on one face of the helix (Figure 2.12B). Because JAZ10.3 lacks L186 (i.e., the protein is truncated after R185), we hypothesized that extending the C -terminus of JA Z10.3 could restore the JAZ10.3 -COI1 interaction. To test this idea, we used site -directed mutagenesis to add a single Leu residue (corresponding to L186) to the C -terminus of JA10.3. The resulting protein (JAZ10.3 +L186 ) was 81! Figure 2.12. JAZ10.3 -COI1 interaction is restored by addition of Leu186 to the C -terminal end of JAZ10.3 (A) Amino acid sequence of JAZ1, JAZ10.1 and JAZ10.3 C -terminus. The hormone -binding loop and Jas motif helix are indicated (Sheard et al. 2010). Leu and Val resi dues that group together to form one face of the helix are highlighted in red. 82!Figure 2.12 (contÕd) . (B) Helical wheel plots of Jas motif helix from JAZ1 and JAZ10. Residues were color -coded based on their physical properties. The three non -polar residues forming a face in the helix (L209, L213 and V220 in JAZ1 and L175, L179 and L186 in JAZ10.3) as well as the last amino acid in JAZ10.3 (R185) are indicated. (C) Coronatine (COR) -mediated JAZ -COI1 interaction in Y2H assays. Yeast strains were co -transformed with COI1 and JAZ10.1, JAZ10.3 or JAZ10.3+ L186 . Yeast colonies were plated on media containing different concentrations of COR, as indicated. As negative and positive controls for protein interaction, yeast was also transformed with empty vector (EV) and JAZ10.1 respectively. (D) In vitro pull -down assays performed with JAZ10.1, JAZ10.3 and JAZ10.3+ L186 . Assays were performed in the presence of difference concentrations of coronatine (COR). Coomassie Blue -stained gel (CB) is shown as a loading control. 83! then evaluated in Y2H assays for ligand -dependent interaction with COI1. The results showed that, in comparison to the full -length JAZ10.1 isoform, COR stimulated very weak interaction of JAZ10.3 with COI1, as previously described (Chung and Howe 2009) ( Figure 2.12C). Strikingly, however, the addition of L186 in JAZ10 +L186 restored this interaction to a level comparable to that of JAZ10.1. In vitro pull -down experiments confirmed that the addition of L186 to JAZ10.3 fully restores ligand -dependent interaction with COI1 (Figure 2.12D). To evaluate whether the strong interac tion of JAZ10.3+L186 with COI1 depends on particular biochemical features of the C -terminal amino acid, we constructed a complete series of JAZ10.3 variants in which L186 was substituted with the remaining 19 individual amino acid residues. The resulting s et of JAZ10.3 derivatives was tested in Y2H system for interaction with COI1 in the presence of COR. The results showed that the addition of positively charged amino acids and most amino acids containing a non -polar or polar uncharged side chain was suffic ient to restore JAZ10.3 -COI1 association in the presence of COR (Figure 2.13 ). However , substitution of L186 with negatively charged or bulky aromatic side chains did not restore interaction with COI1 . To evalua te the relevance of L186 in the function of JAZ10.3, we tested the ability of JAZ10.3 +L186 (expressed from the JAZ10 promoter) to complement the JA -hypersensitive phenotype of jaz10 -1. None of the T2 progeny from eight independent JAZ10p:HA -JAZ10.3 +L186 transgenic lines showed altered sensitivity t o JA in comparison to the parental jaz10 -1 mutant (Figure 2.14A). By contrast, a jaz10 -1 line expressing JAZ10p:HA -JAZ10.3 showed a WT -level of sensitivity to the hormone. In agreement with these findings, Western blot analysis showed that whereas HA -JAZ10 .3 accumulates in a JA -dependent manner in JAZ10p:HA -JAZ10.3 seedlings, JAZ10.3 +L186 does not (Figure 2.14B). These collective findings demonstrate that the 84! Figure 2.13. Complementation of JAZ10.3 -COI1 interaction is dependent on the physical properties of amino acid (aa) residues. Y2H analysis of COI1 interaction with variants of JAZ10.3 in which an extra amino acid residue was added to its C -terminus end. Yeast strains were co -transformed with COI1 and JAZ10.1, JAZ10.3 or a JAZ10.3 derivativ e. Yeast was also transformed with empty vector (EV) and JAZ10.1 As a negative and positive controls for protein interaction, respectively. Coronatine (COR - 100 µM) was used to test for COI interaction. 85! Figure 2.14. JA -stability of JAZ10.3 is cause d by a single amino acid truncation in the C -terminal end of its degron. (A) Root elongation assay Root elongation assay on WT, jaz10 -1 and jaz10 -1 lines transformed with JAZ10p:HA -JAZ10.3 or JAZ10p:HA -JAZ10.3 +L186 . Eight independent JAZ10p: HA-JAZ10.3+ L18 6 lines transgenic lines are shown. Root length was measured in eight -day old seedlings grown in MS medium supplemented or not with 20 µM MeJA. The root length ratio was calculated by dividing the average root length of seedlings grown on medium containing MeJA by the average root length of the same genotype grown in MS medium lacking MeJA. Data show the mean ± S.E. of at least ten seedlings for each genotype. Letters denote a statistical difference in comparison to WT control (TukeyÕs HSD, p-value<0.05). (B) Accumulation of HA -JAZ10.3 protein in seedlings of jaz10 -1 carrying the JAZ10p: HA-JAZ10.3 or JAZ10p: HA-JAZ10.3+L Jas21 transgenes. Two JAZ10p: HA-JAZ10.3+L Jas21 lines (G7 and G17) are shown. Seedlings were grown for eight days in MS media in the absence (-) or presence (+) of 20 µM MeJA. Coomassie blue -stained membrane (CB) is shown as a loading control. 86!removal of L186 via alternative splicing plays a major role in the stability and repressive function of JAZ10.3. JAZ10.3 -like JAZ gene s are widespread i n the plant kingdom Sequence analysis on the JAZ genes of evolutionary diverse plant species highlighted the frequent occurrence of an intron splitting the Jas domain into a two submo tifs organization (Chung et al. 2010). Remarkably, the retention of this conserved intron through alternative splicing can lead to the formation of JAZ variants lacking the last amino acid in their degron sequence in a similar fashion as observed for the JAZ10.3, leading us to speculate that the formation of this type of repre ssors is conserved in the plant kingdom. To gain additional insights into evolutionary and functional significance of the JAZ10.3 -like genes, we searched the Phytozo me genome database for the presence of JAZ -related genes lacking only the last amino acid in their degron sequence. Our analysis showed that the phylogenetically diverse plant species such as rice ( Oryza sativa), orange ( Citrus sinensis), cucumber ( Cucumis sativus), rubber tree ( Hevea brasiliensis ) and others can produce a JAZ10.3 -like protein, where their C -terminal ends in a stop codon truncating the degron in the last amino acid (Table 2.1). This list is just a glimpse of how widely spread this phenomenon is, since the se quencing of new plant genomes and the frequent identification of new splice variants will likely increase the number of examples found. Indeed, it has been experimentally demonstrated that three other Arabidopsis JAZ genes (JAZ2, JAZ6 and JAZ11 ) produce sp lice isoforms similar to JAZ10.3, generating JA -stable JAZ repressors (Chung et al. 2010). These results indicate that the presence of a JA -stable JAZ generated by the absence of the last amino acid in their degron sequence is a common strategy that evolve d the plant kingdom to finely regulate the amplitude and duration of the JA responses. 87! Table 2.1. Examples of JAZ genes missing the last amino acid in the JAZ degron in different plant species a. SPECIES FAMILY LOCUS NAME TIFY SEQUENCE JAZ DEGRON SEQUENCE Oryza sativa Poaceae Os03g27900 TIVYGG VMPIARKASLQRFLQKRKQK* Brachypodium distachyon b Poaceae Bradi3g10820.2 TIFYNG DLPIARKASLHRFLEKRKDR* Citrus sinensis Rutaceae orange1.1g030695 TIFYNG DLPIARRKSLQRFLEKRKER* Arabidopsis thaliana b Brassicaceae At1g74950 (JAZ2) TIFYGG ELPIARRASLHRFLEKRKDR* At1g72450 (JAZ6) TIFFGG VERIARRASLHRFFAKRKDR* At5g13220 (JAZ10) TIFYNG DLPIARRKSLQRFLEKRKER* At3g43440 (JAZ11) TIIFGG DVPIARRRSLQRFFEKRRHR* Capsela rubellata Brassicaceae Carubv10002439 TIFYNG DLPIARRKSLLRFLEKRKER* Brassica rapa Brassicaceae Bra023399 TIFYNG DLPIARRKSLQRFLEKRKER* Malus domestica Rosaceae MDP0000757701 TIFYAG AVPQARKASLARFLEKRKER* MDP0000891920 TIFYAG AVPQARKASLARFLEKRKER* Cucumis sativus Cucurbitaceae Cucsa.095580.1 TIFYNE DLPLARKRSLHRFLEKRKER* Linum usitatissimum Linaceae Lus10002576 TIFYNG ADLPIARRKSLQRFLEKRKER* Hevea brasiliensis c Euphorbiaceae HbJAZ_1660 TIFYAG DLPIARRASLHRFLEKRKDR* HbJAZ_29511 TIFYNG DLPIARRKSLQRFLEKRKER* a- JAZ genes were searched on Phytozome for sequences containing the TIFY (PF06200) and Jas (PF09425) motifs. b- Chung et al. 2010. c- Pirrello et al. 2014. 88!Discussion Splice variants of JAZ10 regulate the amplitude of JA responses Alternative splicing is a widespread mechanism that increases protein diversity and gene function in eukaryotes, providing additional layers of regulat ion in biological networks. It is becoming increasingly evident that alternative splicing is a prevale nt process in plants (Li et al. 2014; Mandadi and Schol thof 2015; Marquez et al. 2012), but examples demonstrating the biological relevance of alternative splicing in plant growth and development is scarce. We focus our work o n the JAZ family of repressors and more specifically on Arabidopsis JAZ10, which is subject to alternative splicing to generate three protein isoforms that differentially interact with COI1 in the presence of t he bioactive JA (Chung and Howe 2009; Chung et al. 2010; Moreno et al. 2013). In an effort to understand the fu nction of these spli ce isoforms, we first evaluated JAZ10 gene expression upon induction by mechanical wounding. Transcripts for all alternatively spliced JAZ10 transcripts were rapidly (<1h ) and st rongly stimulated by leaf injury , in agreement with the observation that this gene is a robust marker for activation of the JA pathway (Mousavi et a l. 2013; Yan et al. 2007). On the other hand, even though splicing can be regulated in a tissue specific manner and dependent on developmental and environmental cues (Pos” et al. 2013; Reddy et al. 2013; Staiger and Brown 2013), we found that JAZ10.1 was consistently the most abundant transcript in all wounded and wounded tissues. Moreover, the relative proportions of the alternatively spliced transcripts derived from JAZ10 pre-mRNA tende d to remain constant in all tissue types and induction conditions studied. These results suggest that the relative abundance of alternative spliced JAZ10 mRNAs is dictated by the strength of splice sites and that the spliceosome components that guide JAZ10 pre -mRNA splicing occur in all tissues. 89! Alterations in the reading frame caused by alternative splicin g can lead to the formation of premature termination codon s (PTC s), which trigger the process of non -sense mediat ed mRNA decay (Wang and Brendel 2006). Even though PTCs are formed in JAZ10.2 and JAZ10.3 , immunoblot experiments indicated that all JAZ10 tran scripts are indeed translated , ruling out the possibility that this form of gene silencing controls JAZ10 expression . Our results indicate that difference s in the strength with which JAZ10 proteins variants interact with COI1 is a much more critical factor in the control of JAZ10 protein levels in stimulated cells. Because mechanical wounding causes a rapid and massive ri se in JA -Ile levels (Koo et al. 2011), JAZ repressors that strongly associate with COI1 in the presence of the JA-Ile will be rapidly targ eted for proteasome -mediated degradation (Thines et al. 2007; Shyu et al. 2012). Consistent with this idea , we found that JAZ10.1, which interacts strongly with COI1 (Chung and Howe 2009), is the most abundant JAZ10 transcript, but the most unstable protei n isoform in wounded leaves . Conversely , the enhanced stability of JAZ10.3 and JAZ10.4 in JA -stimulated cells is a direct consequence of alternative spliced -truncation of the COI -interacting Jas motif. Plant hormones are potent modulators of plant fitness , controlling virtually every aspect of pl ant growth and development (Fonseca et al. 2014b; Nemhauser et al. 2006). Thus, it is not surprising that plants have evolved complex strategies to regulate hormon e biosynthesis and signaling . Among the mechanisms that control the amplitude and duration of JA responses are the catabolism of JA-Ile (Heitz et al. 2012; Koo et al. 2011), the formation of transcriptional regulators that compete with JA -related TFs for DNA binding (Fonseca et al. 2014a; Song et al. 2013) and the formation of stable JAZ repressors (Chun g and Howe 2009; Moreno et al. 2013; Shyu et al. 2012). The observation that the JAZ10.3 is the most abundant JAZ10 isoform in induced cells provided initial evidence that this protein plays a major role in attenuation of JA 90!responses. Complementation experiments performed with the jaz10 -1 null mutant provided definitive evidence for this. These experiments also indicate that JAZ10.4, although present in JA- stimulated cells in relative low levels, also funct ions as a dominant repressor of JA signaling. Our data further suggest any environmental stress capable of activating JA synthesis will lead to de novo synthesis of all three JAZ10 splice isoforms. Under sustained or chronic stress, however, only the stabl e JAZ10.3 and JAZ10.4 repressor will accumulate. Direct interaction of these splice variants with TFs such as MYC will then dampen JA responses. The multiple JA -hypersensitive phenotypes observed in jaz10 -1 are therefore a consequence of the prolonged acti vity of the JA pathway caused by the absence of these stable JAZ 10 repressors. An important question that remains to be addressed is how JAZ10.3 and JAZ10.4 are further removed JA-stimulated cells such that the plants regain its ability to respond robustl y to subsequent stress events that trigger JA production . The existence of a mechanism to remove these stable JAZ repressors is supported by the complete absence of detectable JAZ10.3 and JAZ10.4 protein signal 24hrs after induction of the system (Figure 2 .6A). It is possible that a JA derivative other than JA -Ile acts as a ligand to promote association of these proteins with COI1, although evidence for this hypothesis is currently lacking (Heitz et al. 2012; Koo et al. 2011). Alternatively, stable JAZ repr essors may be targeted for degradation through the action of E3 ligases other than SCF COI1 . Finally, the light -dark cycle may also influence the stability of these proteins, as no protein was detected in the 24 h-time point , which is the only where samples were collected in the absence of light (Figure 2.2B) . Indeed, there is evidence to indicate that JA responses are controlled by the circadian clock (Goodspeed et al. 2012). 91!Integrity of the JAZ degron is necessary for COI1 interaction JAZ proteins contain a degron sequence within the Jas motif that interacts with COI1 in the presence of JA -Ile (Katsir et al. 2008; Melotto et al. 2008; Sheard et al. 2010). It is now evident that natural sequence variation in the degron, together with various mechanis ms to modify the degron sequence generate a repertoire of JAZ variants that differentially interact with COI1 ; as a consequence, plant cells contain multiple JAZ repressors with a wide spectrum of stability at a given concentration of JA -Ile (Chung et al. 2010; Moreno et al. 2013; Shyu et al. 2012). In the case of JAZ10, we introduced a R171A point mutation that impaired ligand -dependent COI1 binding and thus stabilized JAZ10.1. Similar Ala substitutions were previously shown to impede COI1 interaction with JAZ1 and JAZ9 without affecting JAZ interaction with MYC2 (Melotto et al. 2008; Withers et al. 2012 ). Experiments with JAZ10.1 R171A confirmed that this variant behaves as a strong dominant repressor and is capable of reducing the sensitivity of jaz10 -1 to JA. The stabilized JAZ10.1 R171A protein may be a useful tool for further studies of JAZ10 function. A major challenge in the JA field is to determine whether individual JAZ proteins perform different functions. We thus addressed the question of whethe r JAZ8, a nother well -characterized stable JAZ repressor (Shyu et al. 2012), is functionally equivalent to JAZ10. Our strategy was to express JAZ8 under the control of the JAZ10 promoter . This experiment was performed in the jaz10 -1 null genetic background in order to evaluate whether JAZ8 can complement the function of JAZ10 in repressing JA responses. Interestingly, although JAZ8 overexpression can lead to JA insensitivity in roots (Shyu et al. 2012) and that JAZ8 protein was produced in response to JA sti mulation, this stable repressor was unable to complement the JA -hypersensitivity of jaz10 -1. JAZ8 and JAZ10 interact with similar sets of TFs and dimerize with 92!similar JA Z proteins (Chung et al. 2009; Qi et al. 2011) . However, their mechanism of repression of the J A signaling pathway is distinct. JAZ10 interacts with the adaptor protein NINJA to indirectly recruit the co-repressor TOPLESS (TPL) , whereas JAZ8 directly interacts with TPL through an EAR motif located at the N -terminus of JAZ8 (Moreno et al. 20 13; Pauwels et al. 2010; Shyu et al. 2012). The demonstration that NINJA is indispensable for repress ion of JA signalin g in r oots (Acosta et al. 2013) may explain why JAZ8 could not complement the jaz10 -1 root phenotype. Taken together, these observations provide direct genetic evidence for functional specificity among the JAZ family members. We also investigated the mechanism by which alternatively spliced -mediated truncation of JAZ10 increases the stability and repressive activity of JAZ10.3 . We found th at extension of the JAZ10.3 C -terminus by addition of L186 not only restore ligand -dependent interaction with COI1, but also eliminates the repressive function of the protein through destabilization. Structural studies demonstrate that the C -terminal end of the JAZ degron forms an #-helix that serves as a low -affinity anchor that, in the presence of JA -Ile, docks the JAZ protein on COI1 (Sheard et al. 2010). Our results support a model in which altern ative splicing -mediated truncation of JAZ10.3 at R185 removes a key part of the helix (L186) that is required for COI1 interaction, thus stabilizing the protein in the presence of JA. It is also possible that the structure of the helix may be affected by a bsence of L186 It is becoming evident that variation in the JAZ degron sequence is a widespread mechanism to generate stable JAZ repressors that are required for appropriate restraining of JA signaling . JAZ genes in diverse plant species are su bject to a lternative splicing events that modify the degron to create a spectrum of repressors that differentially i nteract with COI1 (Chung et al. 2009). Furthermore, the observation that diverse plant species use alternative 93!splicing to generate stable JAZ10.3 -like repressors highlight the importance of alternative splicing as a fundamental regulatory feature that evolved concomitantly with the appearance of the JA pathway in land plants. Stable JAZ repressor modulate resource allocation To thrive in an ever -cha nging environment, plants need to constantly modify their growth and development to respond to external signals. However limitation s in resource availability may create tradeoffs between growth - and defense - related processes (Herms and Mattson 1992; Huot et al. 2014). Plants appear to use JA as one mechanism to regulate this type of resource allocation . Complex regulatory networks controlling JA biosynthesis and signaling are interconnected with other hormone signaling pathways to presumably op timize plant fitness in changing environments (Ballar” 2011; Erb et al 2012; Yang et al 2012) . Misregulation of the JA pathway leads to a n imbalance in resource al location (Leone et al. 2014; Yan et al. 2007) . For example , strong JA -induced inhibition of r oot growth, increased expression of JA -related genes and insensitivity to FR -light observed in the jaz10 -1 mutant ((Leone et al. 2014) highlight how the absence of JAZ10 repressors lead to over -activation of defense processes and downregulation of growth. In WT plants, JAZ10.3 and JAZ10.4 (and other stable JAZ proteins ) are part of an essential regulatory feedback loop that is activated upon induction of the JA pathway to directly control the activity of TFs. This level of feedback control presumably evolve d as a mechanism to rewires protein -protein interaction networks that serve to fine tune metabolic pathways and the balance between growth and defense processes. 94!Methods Plant material and growth conditions Arabidopsis thaliana plants were grown in so il at 20 +/ - 1¡C under long -day condition s (16h light, 120 µE m-2.s-1). Columbia -0 was used as the wild -type (WT) genetic backgrou nd for all experiments . The jaz10 -1 mutant (SAIL_92_D08; Sehr et al. 2010) was obtained from the Arabidopsis Biological Resource Center. The 35S:JAZ10 G line was des cribed previously (Chung et al. 2010). JA -mediated root growth inhibition assays were performed as previously described (Moreno et al. 2013; Shyu et al. 2012). Unless otherwise noted, all experiments were independently repeated at least three times . Wounding time -course experiment Mechanical wounds were inflicted to leaves of four week -old soil -grown plants with a hemostat as previously described (Koo et al. 200 9) (Figure 2.2) . Wounded leaves were selected on the basis of their position in the st em (Mousavi et al. 2013). Leaf tissue was harvested and immediately frozen in liquid nitrogen prior extraction of RNA or protein (see below). To reduce plant -to-plant var iation, leaves from three plants were pooled for analysis of each time point. Five unwounded systemic leaves were collected an pooled per plant ( Figure 2.2 ). RNA extraction and qRT -PCR Plant tissue was ground to a fine powder and used for RNA extraction with a RNeasy kit (Qiagen) followed by on -column DNase treatment (Qiagen) according to the manufacturerÕs instructions . cDNA was reverse transcribed from 1 µg total RNA with random primers and the High Capacity cDNA Reverse Transcriptio n Kit (Applied Biosy stems, ABI) . qRT -PCR was 95!performed on an ABI 7500 qPCR instrument (ABI), using Power SYBR Green (ABI). Reactions consisted of 2 µL of cDNA template (0.5ng/ µL), 1 µL forward an d reverse primers (5 µM) (Table 2.2), 5 µL of Power SYBR master mix and 2 µL of nu clease-free water . Reactions were incubated under the following conditions: 50 oC for 2min, 95 oC for 10min and 40 cycles consisting of 95 oC for 15s and 60 oC f or 60s. D issociation curve s confirmed primer specificity. No-template controls were included for each primer set to confirm the absence of contamination or primer dimers. Transcript levels for reference genes SERINE/THREONINE PROTEIN PHOSPHATASE 2a (PP2a ) and YELLOW-LEAF-SPECIFIC GENE8 (YLS8 ) ( Vandesompele et al. 20 02) were used to normalize gene expression of all studied genes. All reactions were performed with a minimum of two technical replicates per RNA sample . Protein extraction and immunoblots analysis Protein was extracted from frozen ground tissue by the add ition of 1 mL of lysis buffer (50 mM Tris -HCl, 150 mM NaCl, 1% Triton X -100, 50 µM MG132, 1 µM phenylmethylsulfonyl fluoride , pH8.0 and one tablet of MiniProtean cocktail (Roche) per 10 mL of lysis buffer) per gram of tissue powder. Samples were gently rocked at 4 oC for 10 min and then centrifuged at 14,000g for 15 min at 4 oC. The resulting supernatant was transferred to a new tube and used for protein quantification. Protein samples were resolved on 10% polyacrylamide gels or on 14% Tricine -SDS-PAGE gel s, prepared as described by Sch−gger (2006). I mmunoblots were carried out using an anti -HA antibody ( Covance ) as previously described (Moreno et al. 2013). 96! Table 2.2. List of PCR primers used in this chapter. TARGET NAME PRIMER SEQUENCE NOTES Quantification of transcript levels by qRT -PCR PP2a PP2a_qPCR_Fw 5Õ-AAGCAGCGTAATCGGTAGG -3Õ Described in Attaran et al., 2014 PP2a_qPCR_Rv 5Õ-GCACAGCAATCGGGTATAAAG 3Õ YLS8 YLS8_qPCR_Fw 5Õ-CTCTCAAGGACA AGCAGGAGTTCATT -3Õ Described in Attaran et al., 2014 YLS8_qPCR_Rv 5Õ-CGGTATTTGGTG GAGTAATCTTTTGG -3Õ JAZ10.1 JAZ10.1_qPCR_F w 5Õ-GAAGCGCAAGGAGAGATTAG -3Õ JAZ10.3 JAZ10.3_qPCR_F w 5Õ-AAGGAGAGGTAAT GATTCTTCAACAAT -3Õ JAZ10.1/3 JAZ10.1/3_qPCR_ Rv 5Õ-AGCCAAATCCAAAAACGAACA -3Õ Same Rv primer is used to amplify JAZ10.1 and JAZ10.3 JAZ10.2 JAZ10.2_qPCR_F w 5Õ-CCCCCAAATAATT AAAGAAAGGTTTTT -3Õ JAZ10.2_qPCR_R v 5Õ-AAGCATGTGCGTTGTTGAACA -3Õ JAZ10.4 JAZ10.4_qPCR_F w 5Õ-GCTAATGAAGCAG CATCTAAGAAAGA -3Õ JAZ10.4_qPCR_R v 5Õ-GCGATGGGAAGATCGAAAGA -3Õ OPR3 OPR3_qPCR_Fw 5Õ-GTTACAAGGTGTT AATGGCTCAAAGC -3Õ OPR3_qPCR_Rv 5Õ-ATCACTCCCTTGCCTTCCAGAC -3Õ LOX3 LOX3_qPCR_Fw 5Õ-CCTAGACCGGAT TAATGCGCTAGAC -3Õ LOX3_qPCR_Rv 5Õ-GACCGATGTTTTGGACCATGGGG -3Õ Generation of transgenic lines JAZ10 promoter JAZ10pro NotI GW_Fw 5Õ-CACCGCGGCCGCGA CTTTGGCGAGCAAACC -3Õ Describe in Moreno et al., 2013 JAZ10pro NotI GW_Fw 5Õ-GCGGCCGCCTTCTTTG ATCTTATTAGAAAGTG -3Õ JAZ10g and JAZ10 CDS JAZ10 HA NotI GW_Fw 5Õ-CACCATGTACCCTTATGATGTGCCA GATTATGCCTCTTCGAAAGCTAC -3Õ Describe in Moreno et al., 2013; Fw primer used to amplify JAZ10g and all JAZ10 SV HA-JAZ10g JAZ10g_Rv 5Õ-GTTATAATTTTCTT TACCATATACTAAA -3Õ HA-JAZ10.1 JAZ10.1_Rv 5Õ-TTAGGCCGATGTCGGATAGTAAGG -3Õ HA-JAZ10.3 JAZ10.3_Rv 5Õ-TTACCTCTCCTTGCGCTTCTCGAG -3Õ HA-JAZ10.4 JAZ10.4_Rv 5Õ-CTAATCTCTCCTTGC GCTTCTCGAGAAAACG -3Õ HA-JAZ8 JAZ8_Fw 5Õ-CCACGCGGCCGCATGTACCCTT ATGATGTGCCAGATTATGCCTCT -3Õ JAZ8_Rv 5Õ-TTATCGTCGTGAATGGTAC -3Õ Site -directed mutagenesis (Y2H and transgenic plants) JAZ10.1 -RJas6A JAZ10.1 RJas6A_Fw 5Õ-GATCTTCCCATCGCAGC GAGAAAGTCACTGCAACGT -3Õ Same primer pair is used to perform mutagenesis in all used vectors JAZ10.1 RJas6A_Rv 5Õ-ACGTTGCAGTGACTTTC TCGCTGCGATGGGAAGATC -3Õ 97!Table 2.2 (contÕd). TARGET NAME PRIMER SEQUENCE NOTES Site -directed mutagenesis (Y2H and transgenic plants) JAZ10.3+L JAZ10.3+L_Fw 5Õ-GCAAGGAGAGATTA TAAGGCCGACTCGAGAAG -3Õ JAZ10.3+L_Rv 5Õ-CTTCTCGAGTCGGCC TTATAATCTCTCCTTGC -3Õ JAZ10.3+A JAZ10.3+A_Fw 5Õ-GCAAGGAGAGAGCGT AAGGCCGACTCGAGAAG -3Õ JAZ10.3+A_Rv 5Õ-CTTCTCGAGTCGGCC TTACGCTCTCTCCTTGC -3Õ JAZ10.3+V JAZ10.3+V_Fw 5Õ-GCAAGGAGAGAGTGTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+V_Rv 5Õ-CTTCTCGAGTCGGCC TTACACTCTCTCCTTGC -3Õ JAZ10.3+I JAZ10.3+I_Fw 5Õ-GCAAGGAGAGAATCTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+I_Rv 5Õ-CTTCTCGAGTCGGCCT TAGATTCTCTCCTTGC -3Õ JAZ10.3+P JAZ10.3+P_Fw 5Õ-GCAAGGAGAGACCCTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+P_Rv 5Õ-CTTCTCGAGTCGGCCT TAGGGTCTCTCCTTGC -3Õ JAZ10.3+M JAZ10.3+M_Fw 5Õ-GCAAGGAGAGAATGTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+M_Rv 5Õ-CTTCTCGAGTCGGCCT TACATTCTCTCCTTGC -3Õ JAZ10.3+F JAZ10.3+F_Fw 5Õ-GCAAGGAGAGATTCTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+F_Rv 5Õ-CTTCTCGAGTCGGCCT TAGAATCTCTCCTTGC -3Õ JAZ10.3+W JAZ10.3+W_Fw 5Õ-GCAAGGAGAGATGGTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+W_Rv 5Õ-CTTCTCGAGTCGGCCT TACCATCTCTCCTTGC -3Õ JAZ10.3+G JAZ10.3+G_Fw 5Õ-GCAAGGAGAGAGGGTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+G_Rv 5Õ-CTTCTCGAGTCGGCCT TACCCTCTCTCCTTGC -3Õ JAZ10.3+S JAZ10.3+S_Fw 5Õ-GCAAGGAGAGATCCTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+S_Rv 5Õ-CTTCTCGAGTCGGCCT TAGGATCTCTCCTTGC -3Õ JAZ10.3+T JAZ10.3+T_Fw 5Õ-GCAAGGAGAGAACCTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+T_Rv 5Õ-CTTCTCGAGTCGGCCT TAGGTTCTCTCCTTGC -3Õ JAZ10.3+C JAZ10.3+C_Fw 5Õ-GCAAGGAGAGATGCTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+C_Rv 5Õ-CTTCTCGAGTCGGCCT TAGGATCTCTCCTTGC -3Õ JAZ10.3+N JAZ10.3+N_Fw 5Õ-GCAAGGAGAGAAACTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+N_Rv 5Õ-CTTCTCGAGTCGGCCT TAGTTTCTCTCCTTGC -3Õ JAZ10.3+Q JAZ10.3+Q_Fw 5Õ-GCAAGGAGAGACAGTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+Q_Rv 5Õ-CTTCTCGAGTCGGCCT TACTGTCTCTCCTTGC -3Õ 98!Table 2.2 (contÕd). TARGET NAME PRIMER SEQUENCE NOTES Site -directed mutagenesis (Y2H and transgenic plants) JAZ10.3+Y JAZ10.3+Y_Fw 5Õ-GCAAGGAGAGATACTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+Y_Rv 5Õ-CTTCTCGAGTCGGCCT TAGTATCTCTCCTTGC -3Õ JAZ10.3+D JAZ10.3+D_Fw 5Õ-GCAAGGAGAGAGACTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+D_Rv 5Õ-CTTCTCGAGTCGGCCT TAGTCTCTCTCCTTGC -3Õ JAZ10.3+E JAZ10.3+E_Fw 5Õ-GCAAGGAGAGAGAGTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+E_Rv 5Õ-CTTCTCGAGTCGGCCT TACTCTCTCTCCTTGC -3Õ JAZ10.3+K JAZ10.3+K_Fw 5Õ-GCAAGGAGAGAAAGTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+K_Rv 5Õ-CTTCTCGAGTCGGCCT TACTTTCTCTCCTTGC -3Õ JAZ10.3+R JAZ10.3+R_Fw 5Õ-GCAAGGAGAGAAGGTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+R_Rv 5Õ-CTTCTCGAGTCGGCCT TACCTTCTCTCCTTGC -3Õ JAZ10.3+H JAZ10.3+H_Fw 5Õ-GCAAGGAGAGACACTA AGGCCGACTCGAGAAG -3Õ JAZ10.3+H_Rv 5Õ-CTTCTCGAGTCGGCCT TACTGTCTCTCCTTGC -3Õ pRMG -nMAL JAZ10.3+L Jas21 pRMG -nMAL JAZ10.3+L Jas21 _F 5Õ-GCAAGGAGAGGTTAGT CGAGCACCACCACCAC -3Õ pRMG -nMAL JAZ10.3+L Jas21 _Rv 5Õ-GTGGTGGTGGTGCTC GACTAACCTCTCCTTGC -3Õ JAZ10p :HA -JAZ10.3 +LJas21 JAZ10p :HA -JAZ10.3 +LJas21 _Fw 5Õ-GCAAGGAGAGGTTATA AAAGGGTGGGCGCGCC -3Õ JAZ10p :HA -JAZ10.3 +LJas21 _Rv 5Õ-GGCGCGCCCACCCTT TTATAACCTCTCCTTGC -3Õ 99! Transgene constructs KAPA HIFI Polymerase (Kapa B iosystems) was used for all PCR reactions performed for cloning purposes , as specified by the manufacturer. Primer sets used are listed in Table 2.2. WT lines expressing the JAZ10 genomic sequence ( JAZ10 G) under the control of the CaMV 35S promoter were previously described by Chung et al. (2010). Expression of JAZ10 g, JAZ10 splice variant s and JAZ8 under the control of the native JAZ10 native promoter was performed as prev iously described (Moreno et al. 2013). Briefly, a 2.0 -kb fragment of the JAZ10 promoter (JAZ10p ; Sehr et al., 2010) was PCR amplified from a n XhoI -predigested bacterial artificial chromosome (clone T31B5 ) and cloned into pEntr -D-Topo to generate pEntr -JAZ10p . Primer sets used to amplify JAZ10g (from genomic DNA) and the full -length cDNA for JAZ10.1 , JAZ10.3 , JAZ10.4 and JAZ8 where designed to add a hemagglutinin (HA) -epitope tag on the N -terminus of the proteins. The resulting amplicons were cloned into pEntr -D-Topo and named accordingly ( e.g. pEntr -HA-JAZ10g ). The JAZ10 promoter was then released from pEntr -JAZ10p using NotI and ligated into NotI -linearized pEntr -HA-JAZ10 or pEntr -HA-JAZ8 vectors. A LR -clonase reaction (Invitrogen) was used to transfer the final constructs into the pGWB401 dest ination vector (Nakagawa et al. 2007), which was then used to transform Agrobacterium tumefaciens (strain C58C1). Transgenic Arabidopsis plants were obtained using the floral dip method (Clough and Bent 1998). Seedlings of the transformed lines (T1) were screened on half -strength Murashige and Skoog (MS) agar plates supplemented with 0.8% sucrose (w/w) and kanamycin (50 µg/mL). At least 24 independent T1 lines were transferred to soil. T2 plants carrying a single T-DNA insertion were selected on the basis of the segregation ratios of Kan -resistant to Kan sensitive plants . These lines were further propagated for the identification of homozygous T3 lines. 100!Yeast two -hybrid (Y2H) analysis Y2H assays were perfo rmed with the Matchmaker LexA system (Clontech). Yeast strain EGY48 was used for co -transformation with pGILDA and pB42AD vectors containing the cDNAs for COI1 , JAZ10.1 and JAZ10.3 as described in Chung and Howe (2008). Yeast t ransformant were grown in 3 mL SD-glucose medium (Clontech) supplemented with ÐUra/ -His/-Trp dropout solution , to an OD 600 of 1.0. Cells were recovered by centrifugation at 5000 rpm for 4min and resuspended in 150 µL of distilled water. A total of 15 µl of cell suspension was added to SD -gal/raf (-Ura/ -His/-Trp) inducing medium containing 200 µg/mL of X -gal. In test for JAZ -COI1 inte raction, coronatine (Sigma -Aldrich) was added to the medium before cells were plated. Pictures were taken 48h after incubation of plates at 30 oC. In vitro pull -down assays Pull -down assays were performed using pR MG-nMAL vectors carrying the cDNA for JAZ10.1 and JAZ10.3 (Chung et al, 2010) or mutant derivatives of these proteins (see below). MBP -JAZ-His6 fusion proteins were expressed and purified as previou sly described (Chung et a l. 2010; Shyu et al. 2012). Leaf extracts from an Arabidopsis transgenic line expressing Myc -tagged COI1 were used as a source of COI1 protein (Melotto et al. 2008) and c oronatine (Sigma -Aldrich) was used as a ligand to evaluate JA Z-COI1 interaction. 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PLOS ONE 3: 3e3699. 109! CHAPTER THREE Rewiring of jasmonate and phytochrome B transcriptional networks simultaneously activate plant growth and defense Contributions: The jazQ and jazQ phyB mutant s utilized in this chapter were developed by Dr. Yuki Yoshida, who also performed the hypocotyl elongation assay in monochromatic chambers that lead to identification of the mutation in the PHYB gene in sjq11 . Dalton Oliveira performed the screening for suppressors and enhancers in the EMS -mutagenized population of jazQ. Dr. Georg Jander wa s responsible for quantificatio n of glucosinolates . 110!Abstract In order to thrive in the face of stressful environmental conditions, plants invest resources into the production of defensive traits. These defense responses are energetically demanding, imposi ng on plants a ÒdilemmaÓ to commit limited metabolic resources to growth - or defense -related processes. The regulation of resource allocation tradeoff s has a profound impact on plant biology and ecological relationships, but the molecular mechanisms behind it are still poorly understood. Here , we use a genetic approach to show that transcriptional rewiring of the jasmonate (JA) and phytochrome B (phyB) signaling pathways can uncouple the growth -defense tradeoffs and describe a novel genotype in which both p rocesses are concomitantly upregulated . We show that a high -order mutant (jazQ ) constitutively activated in the JA pathway exhibits constitutive expression of defensive traits , including increased resistance to herbivores attack. As a tradeoff, jazQ plants have reduced stature and other slow -growth phenotypes . Through the use of a genetic suppressor screen, we show that mutation of the gene encoding the red light receptor phyB rescues the slow growth of jazQ without significantly affecting defense traits . W e provide molecular evidence that uncoupling of growth -defense antagonism in jazQ phyB result s from simultaneous activation of MYC2 and PIF transcription factors that promote the expression of defense and growth -related genes, respectively. Our findings su ggest that growth -defense antagonism may not be dictated by constraints on metabolic resources but rather by hard -wired regulatory programs that exert control over resource partitioning in dynamic environments. Our results suggest a novel approach for biot echnological e fforts to produce crop variaties with improved growth and enhanced pest resistance . 111!Introduction Plants exhibit a remarkable degree of developmental plasticity that all ows them to cope with the rapid and ever -changing environmental circumsta nces they experience as consequence of their sessile nature. In response to biotic stress, plants utilize sophisticated de velopmental programs to mount defense s against the attack er. Although essential for survival, these defense responses are resource demanding and may constrain growth processes (Herms and Mattson 1992; Huot et al. 2014). These growth -defense tradeoff s may have profound impact s on plant physiology and ecolog y. Empir ical e vidence from natural plant populations show , for example, that exposure to various environmental cues (such as the presence of herbivores or plant competitors) can lead to tradeoffs in the evolution of growth or defense traits, such that one is usual ly favored in at the expense of the other (Agrawal et al. 2012; Zt et al. 2012). Economically important crops have been bred to maximize growth and yield, which may constrain the expression of defense traits and t hus necessitate the application of pestic ides and fungicides (Herms and Mattson 1992; Strange and Sco tt 2005). In this sense , knowledge of the molecular mechanisms underlying tradeoffs in resource allocation may facilitate the development of crop varieties that combine high yield with increased pest resistance . A t the present, however, these mechanisms remain poorly understood. Jasmonate (JA ) is a lipid -derived plant hormones that regulates responses to a multitude of biotic and abiotic stresses (Campos et al. 2014; Dombrowski 2008; Goodspeed et al. 2012; Li et al. 2004; Howe and Jander 2008; Wasternack and Hause 2013) . JA also controls a wide variety of growth processes , including cell division and expansion, cell differentiation, flower development and senescence (Browse 2008; Li et al. 2004; Pauwels et al. 2008; Wasternack et al. 2013, Yan et al. 2007 ). Given its dual function in the control of growth and defense, JA play s 112!pivotal role in determining how limited resource s are allocated to specific metabolic pathways. Increasing evidence indicates that JA -regulated reprogramming of gene expression serves to redirect resource allocation from primary metabolism and growth to secondary metabolism and defense (Attaran et al. 2014; Baldwin 1998; Campos et al. 2014; Huot et al. 2014). This molecular ÒswitchÓ involves the action of JASMONATE ZIM -domain (JAZ) family of proteins that bi nd to and inhibit transcription factors (TFs) such as MYC2 to promote defense responses . Stress -induced increases in JA levels promote JAZ degradation via th e ubiquitin -proteasome system, allowing MYC2 and related TFs to transcribe genes that confer resistance to her bivore and pathogen attack (Chini et al. 2007; Thines et al. 2007). This model of induced resistance predicts that genetic removal of one or more JAZ repressors will constitutively activate defense responses and likely affect the growth -defense equilibrium . However, apparent genetic redundancy among the 13 members of the JAZ family in Arabidopsis has hindered rigorous testing of this hypothesis (Demianski et al. 2012; Thines et al. 2007 ; Thireault et al. 2015 ). Consistent with its role as in the mod ulation of growth -defense tradeoffs , the JA signaling pathway is tightly integrated within a larger, highly complex regulatory network that orchestrates hormonal control of plant growth and development. Various components of the JA signaling pathway, for example, mediate crosstalk with signal trans duction pathways for gibberellins (Yang et al. 2012) , brassinosteroids (Campos et al. 2009) and other defense hormones such as ethylene (Lorenzo et al. 2002) and salicy lic acid (Thaler et al. 2012) . JA also interact s with signaling pathways associated with light (Moreno et al. 2009) , pathogen perception (Campos et al. 2014) , temperature sensing (Hu et al. 2013) and many others (Wasternack and Hause 2013) . Dissection of the molecular basis of these interactions is providing exciting new insight into how plants finely tune resource allocation in response to changing environment al conditions . In this 113!context, recent evidence highlight s the interaction of JA and light signaling as a key node for regulation of growth -defense tradeoffs (Ballar ” 2014, Leone et al. 2014 ). Phytochromes (phys) are the principal plant photoreceptor s for percepti on of red and far red light and for detecting neighboring plants that complete for photon capture (Casal 2012). Through their ability to sense changes in the re d to far -red (R:FR) ratio of sunlight caused by plant over crowding and shade , the phy receptors modulate the activity of PIF TFs that promote cell extension -type growth; the resulting growth processes allow plants to better compete for light. These so-called shade avoidance responses include stem and hypocotyl elongation, petiole and leaf extension , increased apical dominance and early flowering (Smith and Whitelam 1997) . However, activation of growth processes during shade avoidance responses impairs the p lantÕs ability to mount robust defense responses to pest and pathogen attack (Cerrudo et al. 2012; Moreno et al. 2009). Th ere is increasing evidence to indicate that repression of defense during shade avoidance responses involves the active suppression of the JA signaling pathway, perhaps through increased activity of JAZ repressors (Cerrudo et al. 2012; de Wit et al. 2013; Leone et al. 2014; Mo reno et al. 2009; Robson 2010). Th e observation that phy-impaired mutants display downregulation of JA -dependent defense responses (Cheng et al. 2013; Zhai et al. 2007) , and that JA -induced growth inhibition is associated with suppression of PIF TFs (Yang et al. 2012) suggest a binary model where in which phy -mediated growth and JA -mediated defense s ignaling pathways reciprocally antagonize each other (Ballar ” 2014; Moreno et al. 2009). From an ecological perspective, antagonistic coupling of these two pathways may provide a mechanism to appropriately allocate limited resources , thus optimizing plant fitness in dynamic environments. Here , we describe a genetic approach to uncouple signal antagonism between the JA and phyB pathways , which allowed us to identify unique Arabidopsis genotype in which growth and 114!defense processes are concomitantly upregulated. This was achieved through initial construction of a jaz quintuple (jazQ ) mutant that exhibits constitutive JA -dependent defense responses and slow growth in the absence of JA treatment. We subsequently employed jazQ as a starting point for a genetic suppressor screen aimed at identifying plants that regain growth while maintaining robust defense phenotypes. Characterization of one such suppressor line (sjq11 ) identifi ed the causal mutation as a non-sense mutation in the PHYB gene, which was co nfirmed by genetic reconstitution of a jazQ phyB sextuple mutant that incorporates the phyB -9 reference allele. Genome -wide transcript profiling revealed that genes normally re pressed by phyB and JAZ are concomitantly upregulated in jazQ phyB plants and al so suggest that the unique combination of jazQ and phyB activates new regulatory circuits that are silent in the individual jazQ and phyB parental lines. These results suggest that the growth -defense antagonism is not dictated by constrains on metabolic resources but rather reflect the circuitry of transcriptional programs that have evolved to optimize resource partitioning in dynamic environments. Results The jaz Q quintuple mutant shows hypersensitivity to exogenous JA and constitutive activation of def ense responses Genetic redundancy among the JAZ gene family has hindered efforts to discern the biological relevance of the JAZ proteins as repressors of JA responses . To help overcome this problem, we used transfer -DNA (T -DNA) insertion mutants to construct a jaz quintuple mutant ( jazQ) that is defective in JAZ1, JAZ3, JAZ4, JAZ9 and JAZ10 (Figure 3.1A-B and Methods). These particular members of the JAZ family were selected on the basis of their chromos omal location , phylogenetic relationship within the JAZ family and interaction with common transcriptional re- 115! Figure 3.1. The jaz quintuple (jazQ ) mutant is defective in JAZ1 , JAZ3 , JAZ4 , JAZ9 and JAZ10 . (A) T-DNA lines used for construction of the jazQ mutant. Genomic organization of each JAZ gene is depicted by white and grey boxes, representing UTRs and exons, respectively. The identity and position of the T -DNA insertion is shown. (B) RT -PCR analysis of JAZ gene expression in WT and jazQ . RNA was obtained from seedlings grown for eight days on plates containing 25 µM MeJA. Red arrows in (A) indicate the position of the primers used for the experiment. The ACTIN1 gene ( ACT1 - AT2G37620) was used as a positive control. 116! gulators ( Katsir et al. 200 8; Yang et al. 2012). To test whether simultaneous disruption of these five JAZ genes affect s plant sensitivity to JA , we grew jazQ seedlings o n solid Murashige and Skoog (MS) medium supplemented with 25 µM of methyl JA ( MeJA). As shown in Figure 3.2 , jazQ seedlings exhibit severe JA -induced inhibition of root and shoot growth in comparison to WT. Quantitative analysis showed that JA -induced root growth inhibition was much more pronounced in jazQ than in the jaz10 -1 single mutant, which is known to be JA hypersensi tive (Demianski et al. 2013) or a jaz 3/4/9 triple mutant (Figure 3.2B ). Interestingly, we found that jazQ seedlings develop shorter roots in the absence of exo genous JA (Figure 3.2 A-B), suggesting that JA responses may be constitutively activa ted in this mutant. These results indicate that genetic removal of five JAZ genes results in strong hypersensitivity to JA. We also observed that jazQ plants grown in absence of exogenous JA accumulate anthocyanin pigments in leaf petioles (Figure 3.2D). Quantitative analysis showed that anthocyanin levels in jazQ petioles are nearly five -fold higher than that in WT (Figure s 3.3A -B). To determine whether jazQ plants over -accumulate other JA -regulated metabolites, we also measured the level of glucosinolat es that perform a major role in anti -insect defense (Schweizer et al. 2013). The results showed that jazQ significantly enhance the levels of both aliphatic and indole glucosinolates (Figure 3.3C ). To evaluate the biological relevance of these findings in plant defense against insect herbivory , we performed insect feeding assays with the generalist herbivore Trichoplusia ni (Cabbage looper) . Neonate larvae reared for 10 days on adult jazQ plants gained significantly less weight than larvae feeding on WT (Figure 3.4 A-B). The de creased mass of the T. ni caterpillars grown on jazQ plants was associated with the de creased consumption of leaf tis sue 117! Figure 3.2. jazQ is hypersensitive to exogenous JA. (A) jazQ seedlings are highly senstitive to JA. The photograph show wild -type (WT) and jazQ seedlings grown for eight days in MS medium supplemented or not with 25 µM of MeJA. Scale bar = 1 cm. (B) Root length of WT, jaz10 -1, jaz3/4/9 triple and jazQ mutant seedlings grown for eight days on MS medium supplemented with 5, 10 or 25 µM MeJA. Seedlings were also grown in MS medium not supplemented with MeJA (indicated as 0 µM ) as a control. Data show the mean ± SE (n>12). Asterisks represent statistical d ifference according to Tukey HSD test ( p-value < 0.05). (C-F) Shoot phenotype of WT (C and E) and jazQ (D and F) seedlings grown for 12 d on MS plates without (C and D) or with 25 µM MeJA (E and F). Scale bar = 0.2 cm. 118! Figure 3.3. Constitutive accumulation of secondary metabolites in jazQ . (A-B) Anthocyanin content in WT and jazQ petioles. Pigments were extracted from excised petioles from ten plants. Data represent the mean ± SE . Asterisks (*) represent statistical differences ac cording to StudentÕs T -test ( p-value < 0.05). A photograph of representative pigment extracts obtained for anthocyanin quantification is show in (B). (C) Quantification of indole and aliphatic glucosinolates in WT and jazQ seedlings. Samples were extracte d from seedlings grown on MS media plates for eight days. Data show the mean ± SE. Ten samples (consisting of 50 seedlings each) were used per genotype. Asterisks (*) represent statistical differences according to StudentÕs T -test ( p-value < 0.05). 119! Figure 3.4. jazQ exhibits increased resistance to insect herbivory. (A) Trichoplusia ni weight after feeding on WT and jazQ plants. Neonate caterpillars were reared on genotypes for 10 days. Data represents the mean ± SE (n=12) . Asterisks (*) represent statistical differences according to StudentÕs T -test ( p-value < 0.05). (B) Photograph of representative T. ni larvae recovered from WT and jazQ . Scale bar = 1 cm. (C) Photograph of seven -weeks -old WT and jazQ plants at the end of feeding assay. Scale bar = 2 cm. 120! on this genotype (Figure 3.4 C). These collective results demonstrate a role for JAZ1/3/4/9/10 in the repression of multiple JA -regulated defense processes. The jazQ mutation impedes plant growth In addition to constitutive expression of defense -related traits, soil -grown jazQ plants exhibited several phenotypes indicative of slow growth. The rosette size of jazQ plants prior to flowering was significantly less than that of WT, which was quantitati vely asses sed as a reduction in petiole length, leaf area, number of rosette leaves 21 days after seed sowing an d rosette dry weight (Figure 3.5 A-E). jazQ plants were also delayed in the time to bolting (Figure 3.5 F-H), although the number of rosette leave s at the time of bolting was not different between jazQ and WT (Figure 3.5 I). These findings indicate that the mutant is not impaired in flower meristem formation per se but rather that the genetic removal of multiple JAZ repressors in jazQ results in slow growth of vegetative tissues. JA-response genes are constitutively upregulated in jazQ Our data suggest that resource allocation in jazQ is shifted toward defense at the cost of growth. Because JAZ proteins function as transcriptional repressors, we used RNA sequencing (RNA -seq) to gain additional insight into the growth and defense phenotypes of jazQ . Sequencing of transcripts from WT and jazQ seedl ings grown in the absence of exogenous JA indentified 1098 genes ( p-value < 0.05 according to DESeq statistical package, Anders and Huber 2010, see methods) that were differentially expressed in jazQ . Gene onthology (GO) analysis of the 597 genes upregulat ed in jazQ showed that many of these genes are associated with secondary metabolic pathways (Table 3.1), including the biosynthesis of glucosinolates, phenylpropanoids 121! Figure 3.5. Growth processes are hindered in jazQ . (A) Photograph of 21 days -old WT and jazQ plants. Scale bar = 1 cm. (B) Projected leaf area of WT and jazQ . Data was obtained from 21 days -old rosettes as described in methods. Data show the mean ± SE (n>20). 122!Figure 3.5 (contÕd) . (C) Petiole length of WT and jazQ . Petiole length was meas ured on the third true leaf of 21 days -old plants. Data show the mean ± SE (n=10). (D) Leaf number of WT and jazQ at 21 days. Data show the mean ± SE (n>10). (E) Dry weight of WT and jazQ. Dry weight was measured by freeze -drying the excised rosette of plants grown on soil for a period of 21 days. Data show the mean ± SE (n=10). (F) Bolting time in WT and jazQ . Data shows the mean ± SE (n>12). (G) Photograph of 30 days -old WT and jazQ plants. Scale bar = 2 cm. (H) Number of days to open the first flowe r in WT and jazQ. Data represents the mean ± SE (n>12). (I) Number of rosette leaves at the time of bolting. Data was obtained by counting the number of leaves on the day that a floral meristem was observed. Data shows the mean ± SE (n>12). For all data s hown, asterisks (*) represent statistical differences according to StudentÕs T -test ( p-value < 0.05). 123! Table 3.1. List of selected gene onthology (GO) biological processes upregulated in jazQ . GO-ID DESCRIPTION p-VALUE GO:0019748 Secondary metabolic process 3.23E -27 GO:0019760 Glucosinolate metabolic process 6.39E -22 GO:0009753 Response to jasmonic acid stimulus 2.47E -14 GO:0009611 Response to wounding 1.30E -10 GO:0006952 Defense response 2.12E -10 GO:0009694 Jasmonic acid metabolic process 3.73E -10 GO:0009698 Phenylpropanoid metabolic process 2.74E -08 GO:0010683 Tricyclic triterpenoid metabolic process 2.08E -06 GO:0051554 Flavonol metabolic process 1.19E -04 124! and triterpenoids. Indeed, we found that the vast majority of genes involved in glucosinolates biosynthesis and breakdown are upregulated in jazQ , relative to WT (Figure 3.6). Inside these categories are found transcript s for many well -characterized JA-responsive genes , including ALLENE OXIDE SYNTHASE (AOS), ALLENE OXIDE CYCLASE (AOC), OPC-8:0 COA LIGASE1 (OPCL1 ), VEGETATIVE STORAGE PROTEIN 2 (VSP2 ) and THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2) (Chung et al. 2008), corroborating the hypothesis that JA resp onses are constitutively active in this mutant . Consisten t with this observation , the GO categories ÒDefense responseÓ, ÒJasmonic acid metabolic processÓ and ÒResponse to woundingÓ were significantly enriched in jazQ (Table 3.1). Identification of suppressors and enhancers of jazQ The constitutive JA -response phenotype of jazQ suggested that the genetic ablation of multiple JAZ repressors causes a shift in resource allocation from growth to defense, consistent with the general theory of growth -defe nse antagonism. jazQ therefore provided a new genetic tool to investigate the molecular components involved in growth -defense tradeoff s. For this purpose we mutagenized jazQ seeds with EMS and screened approximately 30,000 soil -grown M2 plants for individuals in which the growth -defense antagonism is ÒuncoupledÓ. Specifically, we looked for mutants that retained constitutive anthocyanin accumulation but whose slow -growth phenotype was suppressed (e.g., reversion to WT -like growth stature). A total o f 34 such lines were identified and categorized as Class I m utants (Figure 3.7 and Table 3.2 ). An example of such line is sjq11 (suppressor of jazQ 11; Figure 3.9 ), which was selected for detailed characterization as described below. 125!Table 3.2. List of su ppressors ( sjq) and enhancers ( ejq) isolated from a M2 population of EMS -mutagenized jazQ seeds. NAME SUPPRESSED jazQ PHENOTYPES ENHANCED jazQ PHENOTYPES ADDITIONAL PHENOTYPES sjq1 Anthocyanin accumulation in young seedlings. sjq2 Late flowering. sjq3 Late flowering, short petiole. sjq4 Late flowering. sjq5 Late flowering. Dwarf. Increased trichome density. sjq6 Short petiole. sjq7 Late flowering. sjq8 Short petiole, late flowering. sjq9 Anthocyanin accumulation. Wide, round leaves. sjq10 Anthocyanin accumulation. sjq11 Short petiole ejq12 Short petiole. ejq13 Anthocyanin accumulation. Lanceolate leaf shape. ejq14 Anthocyanin accumulation, short petiole, late flowering. Flat leaves. ejq15 Anthocyanin accumulation. Anthocyanin accumulation. ejq16 Anthocyanin accumulation, dwarf sjq17 Short petiole. Increased leaf number at flowering, large siliques. sjq18 Late flowering. sjq19 Short petiole. ejq20 Short petiole (much shorter). sjq21 Anthocyanin accumulation. Dentate leaf shape. sjq22 Short petiole, late flowering. sjq23 Short petiole, anthocyanin accumulation. Late flowering. sjq24 Anthocyanin accumulation. Leaf shape (lanceolate, dentate), smaller plant stature, Increased trichome density. sjq25 Short petiole, late flowering, anthocyanin accumulation (late). ejq26 Anthocyanin accumulation, short petiole. sjq27 Short petiole, late flowering, anthocyanin accumulation (late). ejq28 Dwarf, anthocyanin accumulation. sjq29 Anthocyanin accumulation in young seedlings. sjq30 Short petiole. Curled leaves. ejq31 Anthocyanin accumulation, small rosette, late flowering. Increased trichome density. sjq32 Short petiole. Short petiole, late flowering. sjq33 Late flowering, short petiole (early). 126!Table 3.2 (contÕd). NAME SUPPRESSED jazQ PHENOTYPES ENHANCED jazQ PHENOTYPES ADDITIONAL PHENOTYPES sjq34 Late flowering. Short petiole, late flowering. Curled leaves. sjq35 Short petiole. Anthocyanin accumulation, late flowering. Flat leaves. ejq36 Anthocyanin accumulation, short petiole. Flat leaves. ejq37 Short petiole, late flowering. Anthocyanin accumulation, short petiole. sjq38 Anthocyanin accumulation, short petiole, late flowering. Dentate leaf shape, curled leaves. sjq39 Late flowering, anthocyanin accumulation, short petiole. Curled leaves. sjq40 Short petiole, late flowering. sjq41 Anthocyanin accumulation, short petiole, late flowering. Seeds have greenish color, large wide leaves. sjq42 Anthocyanin accumulation; however, M3 plants accumulate some anthocyanins, have increased trichome density. Short petiole, late flowering. ejq43 Anth accumulation, short petiole. Increased trichome density. ejq44 Short petiole, anthocyanin accumulation, late flowering. ejq45 Short petiole, anthocyanin accumulation, late flowering. sjq46 Short petiole, late flowering. sjq47 Anthocyanin accumulation. Short petiole, late flowering. Dentate leaf shape; small, flat leaves. sjq48 Short petiole. Short petiole, late flowering. Curled leaves. ejq49 Short petiole, anthocyanin accumulation, late flowering. sjq50 Short petiole, late flowering. sjq51 Short petiole, late flowering. sjq52 Short petiole. Late flowering. ejq53 Anthocyanin accumulation. sjq54 Short petiole, late flowering. Some chlorosis; variable phenotypes. sjq55 Short petiole, anthocyanin accumulation, late flowering. ejq56 Anthocyanin accumulation. Wavy leaves; highly variable phenotypes. ejq57 Anthocyanin accumulation, late flowering. sjq58 Short petiole. ejq59 Short petiole, late flowering. Enhanced jazQ phenotypes. Flat leaves. sjq60 Short petiole. sjq61 Short petiole, late flowering. Short petiole. ejq62 Anthocyanin accumulation. ejq63 Late flowering. Anthocyanin accumulation, short petiole. Lanceolate leaf shape, altered leaf angle, pale -green leaves. 127!Table 3.2 (contÕd). NAME SUPPRESSED jazQ PHENOTYPES ENHANCED jazQ PHENOTYPES ADDITIONAL PHENOTYPES ejq64 Short petiole, late flowering. Ovate leaf shape, flat leaves. sjq65 Late flowering, short petiole. sjq66 Anthocyanin accumulation, short petiole, late flowering. sjq67 Late flowering. Wide leaves. sjq68 Short petiole. Late flowering. Flat leaves, delayed leaf growth. sjq69 Short petiole. Curled leaves. sjq70 Short petiole. sjq71 Short petiole, anthocyanin accumulation. Late flowering. Curled leaves. sjq72 Short petiole. sjq73 Short petiole. 128! Figure 3.6. Genes involved in glucosinolate biosynthesis and breakdown are upregulated in jazQ. Full transcriptome -sequencing in WT and jazQ seedlings show that the majority of the genes involved with GS biosynthesis, including the transcription factors associated with GS production), and GS breakdown are upregulated in jazQ . 129! Figure 3.7. Isolation of enhancers and suppressor mutants from a population of EMS -mutagenized jazQ M2 plants . Approximately 30,000 plants were screened in the M2 generation and four phenotypic classes of mutants were identified: Classes I to III ( suppressors of jazQ Ð sjq) suppress one of jazQ phenotypes. Class I mut ants have a WT -like growth pattern but maintain anthocyanin accumulation in the petiole as jazQ . Class II mutants grow and accumulate anthocyanins as WT whereas class III mutants grow as jazQ but accumulate anthocyanins as WT. Class IV (enhancers of jazQ Ð ejq) mutants show enhancement of a jazQ phenotype. False purple coloration was added to plants to facilitate description. 130! We also ident ified a distinct group of mutants , in which the ant hocyanin accumulation phenotype was suppressed concomitantly or not with suppression of slow -growth (respectively Class es II and III - Figure 3.7). Among the lines where both anthocyanin accumulation and slow -growth was suppressed (Class II ), are included two male sterile plants sjq10 and sjq66 (Figur e 3.8A), which resemble Arabidopsis mutants defective in JA biosynthesis or signaling (Wasternack et al. 2013; Xie et al. 1998). Root inhibition assays showed that sjq10 is fully sensitive to exogenous MeJA, whereas sjq66 is strongly insensitive to the hor mone (data not shown). Subsequent DNA sequencing of candidate genes identified a C !T non -sense mutation in the codon 56 of AOS gene in sjq10 and a C !T missense mutation in the codon 86 of the COI1 gene of sjq66 (Figure 3.8B). These results suggest that the growth and defense phenotypes of jazQ are dependent on functional JA biosynthesis and signaling pathways. Finally, we also identified several mutants in which phenotypes of jazQ are enhanced (Class IV Ð Figure 3.7). These enhancers of jazQ (ejq) lines e xhibited severe dwarfism, increased anthocyanin content and/or delayed flowering. A total of 22 ejq mutants were identified, but there were not further investigated. sjq11 carries a nonsense mutation in the PHYTOCHROME B gene Phenotypes observed in the sjq11 M2 plants, including long petioles, early flowering time (i.e. days to bolting), were confirmed in the M 3 generation (Figures 3.10A -B). Root growth inhibition assays further showed that sjq11 maintains hypersensitivity to JA and also exhibits the con stitutive short root phenotype of parental jazQ mutant (Figure 3.10C). During the course of these experiments, we observed that sjq11 seedlings grown in constant white light have phenotypes reminiscent of photomorphogenic mutants, including elongated hypoc otyls and pale 131! Figure 3.8. sjq66 carries a mutation in the CORONATINE INSENSITIVE1 gene . (A) Photograph of five weeks -old sjq66 plants. Scale bar = 1 cm. (B) Schematic representation of the CORONATINE INSENSITIVE1 (COI1) gene. Genomic DNA was extracted from sjq66 and used for sequence analysis. The cytosine to thymine transition mutation in the COI1 gene of sjq66 is illustrated . 132! Figure 3.9. sjq11 suppresses the slow -growth phenotype of jazQ but not its anthocyanin accumulation in petioles . Photograph of five week -old WT, jazQ and sjq11 plants. Scale bar = 1 cm. 133! Figure 3.10. sjq11 shows improved growth and retains hypersensitivity to JA treatment . (A) Petiole length of WT, jazQ and sjq11 . Petiole length was measured on the third true leaf of 21 days -old plants. Data show the mean ± SE (n=10). (B) Number of days to bolt in WT, jazQ and sjq11 . Data shows the mean ± SE (n>15). (C) Root length of WT, jazQ and sjq11 grown on MS medium supple mented or not with 20 µM MeJA. Data show the mean ± SE (n>12). For all data shown, letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). 134! green cotyledons (Figure 3.11A -B) (Mur amoto et al. 1999; Reed et al. 1994) . To test the hypothesis that sjq11 is impaired in light signaling, we compared the hypocotyl response of sjq11 to the well -characterized photoreceptor mutants grown in monochromatic light chambers that provide specific wavelengths of light (See methods). We found that sjq11 is as insensitive to red light as determined a phyB null mutant ( phyB -9) (Figure 3.12). This hypocotyl response phenotype was specific to red light, suggesting that sjq11 is defective in the phyB red light signaling pathway . Indeed, seq uencing of PHYB gene (AT2G18790) in sjq11 revealed a C !T transition (Figure 3.13) that creates a stop codon in the chromophore -binding domain of the protein. Allelism tests performed with the phyB -9 mutant showed that the F1 plants obtained from a cross be tween sjq11 and phyB-9 display long hypocotyls under white light (Figure 3.14). These findings demonstrate that sjq11 harbors a null mutation in the PHYB gene. jazQ phyB is upregulated in growth and defense parameters. To address the possibility that spurious EMS -induced mutations contribute to phenotypes of sjq11 , we reconstituted a pure jazQ phyB line through a cross between jazQ and phyB -9, followed by selection of a mutant that is homozygous for jazQ and phyB . As was observed in sjq11 , the resulting jazQ phyB sextuple mutant had a larger rosette diameter and petioles with high anthocyanin content (Figure 3.16A -C). The larger rosette diameter of jazQ phyB was attributed in part to longer petioles, which is a hallmark of phyB mut ants (Figure 3.16A). However, the projected leaf area of jazQ phyB was also greater than that of jazQ, WT and phyB plants as well (Figure 3.16B). Despite large differences in rosette diameter and projected leaf area between jazQ and phyB , the rosette dry m ass of these two lines was not significantly different (Figure 3.16C), presumably because of differences in specific leaf area (leaf area/lead 135! Figure 3.11. sjq11 seedlings develop long hypocotyls under white light. (A) Photograph of WT, jazQ and sjq11 seedlings grown on MS medium for eight days, under continuous white light. Representative seedlings of each genotype are shown. Scale bar = 0.2 cm. (B) Hypocotyl length of WT, jazQ and sjq11. Hypocotyls were measured on seedlings grown on MS medium for eig ht days, under continuous white light. Data shows the mean ± SE (n>20). Letters represent statistical differences according to Tukey HSD -test ( p-value < 0.05). 136! Figure 3.12. sjq11 is impaired in red light perception. Hypocotyl elongation in response to different light wavelengths. Seedlings of WT , jazQ , sjq11 and mutants impaired in red ( phyB -9) far -red ( phyA -75) or blue ( cry1-400) light perception were grown for three days on MS medium in monochromatic light. Seedlings were also grown in the dark as a control. Data represent the mean ± SE (n>20). Letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). 137! Figure 3.13. sjq11 harbors a mutation in the PHYTOCHROME B (phyB ) gene. Schematic representation of the phyB gene in sjq11 . Genomic DNA was extracted from sjq11 and used for sequence analysis. Red letters indicate the cytosine to thymine transition that leads to a nonsense mutation (R322*) . 138! Figure 3.14. Genetic non -complementation of log hypocotyl phenotype in sjq11 and phyB -9. Photograph of representative seedlings of WT, jazQ , sjq11 , phyB -9 and the F1 generation obtained from a cross between sjq11 and phyB -9. Seedlings were grown for three days under constant white light. Scale bar = 0.2cm. 139! Figure 3.15. jazQ phyB plants combine the stronger anthocyanin accumulation of jazQ with the large rosette size of phyB -9. (A) Photograph of four weeks -old WT, jazQ , phyB -9 and jazQ phyB plants. Scale bar = 1 cm. (B) Anthocyanin content in WT, jazQ, phyB-9 and jazQ phyB. Pigments were extracted from leaf petioles of 21 days -old plants. Data represent the mean ± SE (n>10). (C) Rosette diameter of WT, jazQ , phyB -9 and jazQ phyB . Data was obtained through image analysis of four week -old plants using ImageJ. Data show the mean ± SE (n>20). For all data shown, letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). 140! Figure 3.16. Growth parameters are improved in jazQ phyB . (A) Petioles length of WT, jazQ , phyB -9 and jazQ phyB plants. Petiole length was measured on the third true leaf of 21 days -old plants. Data represent the mean ± SE (n=10). (B) Projected leaf area of WT, jazQ, phyB-9 and jazQ phyB. Data was obtained from 21 days -old rosettes as described in methods. Data sho w the mean ± SE (n>20). 141!Figure 3.16 (contÕd) . (C) Dry weight of WT, jazQ , phyB -9 and jazQ phyB. Dry weight was measured by freeze -drying the excised rosette of plants grown on soil for a period of 21 days. Data represents the mean (n=10) ± SE. For all dat a shown, letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). (D) Bolting time in WT, jazQ , phyB -9 and jazQ phyB . Data shows the mean ± SE (n>12). (E) Number of leaves at time of bolting in WT, jazQ, phyB-9 and jazQ phyB. Data was obtained by counting the number of leaves on the day that a floral meristem was observed. Data shows the mean ± SE (n>12). (F) Number of days to open the first flower in WT, jazQ, phyB-9 and jazQ phyB. Data represents the mean ± SE (n>12). 142! dry mass). The dry weight of jazQ phyB rosettes was comparable to that of WT and, remarkably, nearly twice that of either jazQ or phyB -9. As in the case for the phyB -9 mutant (Reed et al. 1993), jazQ phyB showed accelerated flowering as determined by measurements of time to bolting and time to opening of the first flower (Figure 3.16D -F). We conclude that loss -of-function of phyB suppresses numerous aspects of the slow -growth phenotype of jazQ . We next evaluated JA - and defense -relat ed traits in jazQ phyB . Root growth inhibition assays showed that, as observed for sjq11 , jazQ phyB seedlings retain both the hypersensitivy to exogenous JA and the constitutive short root phenotype of jazQ (Figure 3.17A). The root length of phyB seedlings was similar to that of WT both in the presence and absence of JA. We also found that jazQ phyB was similar to the jazQ parental line in having small but significant increases in the content of indole and aliphatic glucosinolates, as compared to WT and phyB-9 (Figure 3.17B). Insect feeding assays performed with T. ni larvae provided additional evidence that the robust JA -mediated defense responses exhibited by jazQ are maintained in jazQ phyB. T. ni weight gain on jazQ phyB was similar to that on jazQ and l ess than half of that observed on WT plants (Figure 3.18A -B). There also appeared to be more leaf damage on WT than jazQ and jazQ phyB rosette leaves after 10 days of feeding (Figure 3.18C). We also found that the phyB-9 mutant is extremely susceptible to insect herbivory, consistent with previous studies (Moreno et al. 2009). Feeding trials involving phyB-9 plants had to be terminated early (i.e., within five days of challenge) because of near -complete consumption of phyB -9 leaves, which was accompanied by high weight gain of T. ni larvae reared on this mutant relative to other genotypes (Figure 3.19A-C). This result can be correlated with the lower levels of indole GS in this mutant (Fig ure 3.17B) since these compounds are k nown to play a fundamental role in defense against herbivor es (Hopkins et al. 2009; Schweizer et al. 2013 ). 143! Figure 3.17. jazQ phyB is hypersensitive to JA and accumulates more glucosinolates than WT. (A) Root length of WT, jazQ , phyB -9 and jazQ phyB seedlings grown for eight days in MS medium supplemented or not with 20 µM MeJA. Data show the mean ± SE (n>12). Letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). (B) Glucosinolate content of WT, jazQ, phyB-9 and jazQ phyB. Data show the mean ± SE of ten samples per genotype . Letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). 144! Figure 3.18. jazQ phyB is more resistant to insect herbivory. (A) Trichoplusia ni weight after feeding on WT, jazQ and jazQ phyB plants. Neonate caterpillars were reared on genotypes for 10 days. Data represent the mean ± SE (n=12) . Letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). (B) Photograph of representative T. ni larvae recovered from WT, jazQ and jaz phyB after feeding period. Scale bar = 1 cm. (C) Photograph of seven -weeks -old WT, jazQ and jazQ phyB plants at the end of feeding assay. Scale bar = 3 cm. 145! Figure 3.19. phyB -9 plants are extremely susceptible to insect herbivory. (A) Trichoplusia ni weight after feeding on WT, jazQ , phyB -9 and jazQ phyB plants. Neonate caterpillars were reared on genotypes for five days. The experiment was halted at this time due to full consumption of leaf material in the phyB -9 plants (C). Data represent the mean ± SE (WT, jazQ and jazQ phyB n=4; phyB -9 n=12). Letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). (B) Photograph of representative T. ni larvae recovered from WT, jazQ , phyB -9 and jaz phyB after feeding period. Scale bar = 1 cm. (C) Photograph of six -weeks -old WT, jazQ, phyB -9 and jazQ phyB plants at the end of feeding assay. Scale bar = 3 cm. 146! Rewiring of t ranscriptional networks upregulate growth and defense i n jazQ phyB Results described above suggest two different patterns of resource allocation in the parental jazQ and phyB -9 mutants. The removal of five JAZ genes leads to c onstitutive activation of JA -mediated defense responses but hindered growth in jazQ . On the o ther hand, impaired red light perception results in increased growth parameters at the cost of defense in phyB -9. The combination of jazQ and phyB -9 appears to create a genetic background (jazQ phyB ) in which both robust defense and growth are maintained. Given the direct role of JAZs and phyB in transcriptional control (Chen and Chory 2011; Jiao et al. 2007; Pauwels et al. 2008), we used RNA -seq to test the hypothesis that the uncoupling of growth -defense antagonism in jazQ phyB results from genome -wide re -programmi ng of gene expression. Analysis of RNA -seq data from WT and mutant seedlings grown under identical conditions showed that the overall gene expression pattern of jazQ phyB represents the additive effect of defense processes that are activated in jazQ and t he growth processes that are transcriptionally activated in phyB-9 (Figure 3.20). A comparison of GO categories that are upregulated in both jazQ and jazQ phyB , for instance, identified defense -associated processes such as ÒSecondary metabolism Ó, ÒJA biosynthesis Ó and ÒResponse to wounding Ó (Fig ure 3.20 , blue sector ). As shown in Figure 3.21, genes involved with GS biosynthesis provide an example of a process that is upregulated both in jazQ and jazQ phyB. These results are consistent with the increase d secondary metabolite content and enhanced resistance of jazQ and jazQ phyB (Figure 3.17 to 3.19). In agreement with the well-described antagonism between JA and the defense hormone salicylic acid (SA) (Robert -Seilaniantz et al. 2011), we found that SA re sponses are downregulated in jazQ and jazQ phyB (Figure 3.22, blue sector). 147! Figure 3.20. The combination of phyB -9 and jazQ leads to additive transcriptional effects in jazQ phyB . Venn diagram showing number of upregulated genes in jazQ , phyB -9 and jazQ phyB when compared to WT (Col -0). Gene ontology (GO) analysis indicates that jazQ phyB reflects the additive upregulated defense processes of jazQ (blue region) and the growth processes of phyB (pale-green region). The combination of jazQ and phyB mutations also leads to a reprogramming of processes that are specific to jazQ phyB (red region). Differentially expressed genes were called on the basis of a p-value <0.05 using the statistical package DESeq. 148! Figure 3.21. Genes associated with gluco sinolate biosynthesis are upregulated in jazQ and jazQ phyB but partially downregulated in phyB -9. 149!Figure 3.21 (contÕd) . Heat map showing the expression levels of genes involved in GS biosynthesis in jazQ, phyB-9 and jazQ phyB. Genes were organized according to Sonderby et al. (2010). Values obtained by RNA -Seq represent fold changes (Log 2) over WT. 150! Figure 3.22. The combination of phyB -9 and jazQ leads to additive and synergistic transcriptional reprogramming in jazQ phyB . Venn diagram showing t he number of downregulated genes in jazQ , phyB -9 and jazQ phyB when compared to WT (Col -0). GO analysis was performed with differentially expressed genes and called on the basis of p-value of <0.05 using the statistical package DESeq. 151! A comparison of transcript profiles in phyB -9 and jazQ phyB revealed that these two genotypes share upregulated GO categories related to growth, including ÒResponse to auxin stimulusÓ, ÒShade avoidanceÓ, ÒResponse to red or far -red lightÓ and ÒCell growthÓ (Figure 3.17, pale -green sector). Among the specific upregulated genes are members of the expansin family , including the EXPA3 (AT2G37640), EXPA5 (AT3G29030) and EXPA14 (AT5G56320) , which are involved with cell enlargem ent and growth (Cosgrove et al. 2000 ). We also observed that genes encoding TFs belonging to the PHYTOCHROME -INTERACTING FACTOR (PIF) family such as PIF3 -LIKE1 (PIL1 , AT2G46970) and REDUCED PHYTOCHROME SIGNALING1 (REP1, AT1G02340) are also strongly upregulated in phyB -9 and jazQ phyB . These TFs are required for growth promotion during shade avo idance responses, which is mediated in large part by light conditions (low R:FR ratio) that decreases the activity of phyB (Lorrain et al. 2008). Analysis of gene expression profile in jazQ phyB also revealed a large number of differentially regulated genes that were unique to this genotype (Figure 3.20 red zone). Among the genes that were uniquely upregulated in jazQ phyB were members of the expansin, extensin , pectinase and cel lul ase families involve d in cell wall organization. The GO term ÒCell wall organization or biogenesisÓ was also upregulated in jazQ phyB and included POLTERGEIST LIKE1 (AT2G35350 ), GLABRA3 (AT5G41315 ) and ROTUNDIFOLIA3 (AT4G36380) that are involved in the control of meristem siz e and organ formation (Bancos et al. 2002; Cho and Cosgrove 2000). 152!Overexpression of PHYTOCHROME INTERACTING FACTOR 4 in jazQ partially recapitulates the jazQ phyB phenotype A central component of the phytochrome s transcriptional network are the basic helix -loop -helix (bHLH) PHYTOCHROME INTERACTING FACTOR s (P IFs), TFs that promote a wide range of growth processes, including hypocotyl elongation, cell expansion, chloroplast differentiation and flowering (Chen and Chori 2011; Lorrain et al. 2008; Lucas and Prat 2014). Under light conditions indicative of shade or plant crowding (low R:FR ratios), however, phyB activity is reduced, thus allowing PIF to activate the expression of growth -related genes involved in shade avoidance responses (Leivar and Quail 2011; Lorrain et al. 2008; Lucas and Prat 2014 ; Park et al. 2012). PIF protein s also accumulate in phyB mutants, which display shade avoidance -like growth phenotypes (Park et al. 2004 ; Park et al. 2012 ; Soy et al. 2012 ). The observation that PIF overexpression partially restores JA -induced growth inhibition , together with the ability of JAZs to negatively regulate PIF stability (Yang et al. 2012), lead us to test the hypothesis that the enhanced growth stature of jazQ phyB involves increased activity of PIF4. To t est this, we analyzed the effects of PIF4 overexpression on the growth and defense phenotypes of jazQ . In two independent transgenic line s analyzed (#1 -2 and #3 -1), we found that jazQ plants expressing PIF4 under the CaMV 35S promoter (35S:PIF4 jazQ ) have rosettes that are c omparable in size to WT plants (Figure 3.23 A-B). Petioles of both lines, however, accumulated anthocyanins to similar levels observed in jazQ (Figure 3.23 C). Furthermore, insect feeding assays showed that T. ni larvae reared on 35S:PIF4 jazQ plants gain less weight than larvae reared on WT , similar to the resistance observed for jazQ (Figure 3.24 A-B). Based on these findi ngs, we conclude that overexpression of PIF4 in jazQ background rescues at least 153! Figure 3.23. Overexpression of PIF4 in jazQ leads to partial rescue of growth without affecting defense. (A) Photograph of representative 21 days -old WT, jazQ, phyB-9, jazQ phyB and a representative line of jazQ overexpressing PIF4 (#3-1). Scale bar = 1 cm. (B) Rosette diameter of WT, jazQ, phyB-9, jazQ phyB and two independent 35S:PIF4 jazQ lines (#1-2 and #3 -1). Bars represent the mean ± SE (n>20). Letters indicate st atistical differences according to Tukey HSD -test ( p-value < 0.05). (C) Anthocyanin content in petioles of WT, jazQ, phyB-9, jazQ phyB and the two 35S:PIF4 jazQ lines (#1 -2 and #3 -1). Pigments were extracted from leaf petioles of 21days -old plants. Data represent the mean ± SE (n>10). Letters indicate statistical differences according to Tukey HSD -test ( p-value < 0.05). 154! Figure 3.24. Overexpression of PIF4 in jazQ does compromise resistance to insect herbivory. (A) Trichoplusia ni larvae weight after feeding on WT, jazQ and two 35S:PIF4 jazQ lines. T. ni neonate caterpillars were reared on six week -old for a period of 10 days. Data show the mean ± SE (n=12). Letters indicate statistical differences according to Tukey HSD -test ( p-val ue < 0.05). (B) Photograph of representative T. ni larvae recovered from WT, jazQ and the two jazQ 35S:PIF4 transgenic lines (#1 -2 and #3 -1) after 10 days of feeding. Scale bar = 1 cm. 155! some aspects of the slow -growth phenotype (e.g. petiole extension), but does not compromise the enhanced resistance of jazQ to insect feeding. Discussion The ability to perceive, integrate and trigger proper responses to surroundings signals is a n essential feature for any living organism to prosper in their environment. As sessile organisms, plants utilize their developmental plasticity to respond to adverse conditions , shaping their phenotype in response to the most variable external cues. The growth versus defense paradigm postulates that developm ent al plasticity is limited by resource availability, thus resource allocation to growth will come at the detriment of defense and vice -versa. P oor knowledge of the molecular players and mechanisms controlling resource allocation decisions has impeded furt her understanding on how plants adapt their metabolic fluxes to respond to their surroundings. However , r ecent empirical lines of evidence suggest s that intricate regulatory circuits have evolved to finely tune the growth versus defense balance in response to dynamic environments (Chen et al. 2013; Denanc ” et al. 2013; de Wit et al. 2013; Huot et al. 2014 , Leone et al. 2014; Moreno et al. 2009; Wild et al. 2012; Yang et al. 2012 ). We demonstrate the relevance of the JA -phytochrome B module as a master regul ator o f resource allocation decisions. Impairment of key signaling components belonging to this module altered a vast array of processes at the transcriptional, metabolic and morphological level, leading to a compromise in the balanc e between growth and de fense . Surprisingly, thro ugh genetic manipulation we were able to uncouple the JA -phythochrome B module and activate growth and defense simultaneously . Our results suggest that the growth versus defense duality is not controlled by resource availability 156!per se , but rather by hard -wired transcriptional networks that evolved to precisely modulate the developmental plasticity in response to an ever -changing environment. JAZ proteins inhibit defense and promote plant growth Our starting point to better understand the growth versus defense paradigm was to obtain a plant that was clearly shifted in resource allocation decisions. For this purpose we focused on JAs, lipid -derived plant hormones that are largely characterized with respect to their involvement in defense processes (Campos et al. 2014; Dombrowski 2008; Goodspeed et al. 2012; Li et al. 2004; Howe and Jander 2008; Wasternack and Hause 2013). Studies on the JA signaling pathway have been partially hindered by the functi onal redundancy among the JAZ family of repressors. However, b y genetically knocking out multiple JAZ genes , we were able to generate a mutant that is hypersensitive to exogenous JA treatment, upregulated in the expression of defense related genes and accumulation of secondary metabolites and finally, mo re resistant to insect herbivory . Even though other jaz single mutants have demonstrated some degree of JA hypersensitivity (Demianski et al. 2012; Grunewald et al. 2009; Sehr et al. 2010), jazQ is, to our knowledge, the first jaz mutant described where JA responses are constitutively active and defense processes are clearly upregulated without the need of exogenous hormone treatment. Our findings that growth parameters such as rosette growth and floweri ng time are hindered in jazQ also highlights the tradeoff caused by the constitutive activation of the JA -pathway and the increased allocation of resources in defens ive traits. Eight functional JAZ genes are still present in the jazQ genome and further knockout of those can not only promote the intensification of the se growth and defense parameters , but also lead to the discovery of distinct plant phenotypes 157!that may designate novel biological roles performed by the JAZ repressors (and the JA pathway itself). The shift in the resource allocation decisions described for jazQ is in agreement with numerous described roles of JAs in plant development. For example, the higher anthocyanin and GS content in jazQ correlates with the described role of JA as a positively regulator of the biosynthesis of these secondary metabolites ( Browse and Howe 2008; De Geyter et al. 2012; Qi et al. 2011; Shan et al . 2009; Schweizer et al. 2013). I t is also known that the JAZ repressors can physically interact with and inhibi t the action of TFs involved with anthocyanin and GS production ( Fern⁄ndez -Calvo et al. 2011; Qi et al. 2011; Schweizer et al. 2013). The expression of defense related genes such as AOS, AOC, MYC and TGG2, which are found to be upregulated in jazQ , is also under control of the JA pathway (Attaran et al. 2014; Chung et al. 2008). Recent evidences also indicate that the JAZ proteins can associate with different transcriptional modulators o f growth -related pathways. One e xample is the DELLA, which act as repressors of numerous growth -related processes (Leone et al. 2014; Wild et al. 2012; Yang et al. 2012). DELLA interacts with PIFs, for example, to impede these TFs to promote the expression of growth -related genes. According to proposed models (Kazan and Manners 2012; Yang et al. 2012), JAZ removal by JA -mediated degradation (or by genetic manipulation as in jazQ ) would disrupt the JAZ -DELLA interaction, releasing the DELLAs to further associate with PIFs (and other growth related TF s) and inhibit the activation of growth processes . In conclusion, though a genetic manipulation that knocked out five JAZ genes from a plantÕs genome, we were able to overcome the functional redundancy among the JAZ family members and obtain a genotype that is shifted in th e allocation of resources to growth and defense when compared to WT plants. The downregulation of growth processes caused by the 158!absence of multiple the JAZ genes indicate that they are essential promoters of plant growth, performing this function by inhibiting the allocation of resources to defense responses through repression of the JA signaling pathway. Suppressors of jazQ identify new components involved with resource allocation decisions In an attempt to isolate key components involved in resource allocation decisions, we EMS-mutagenized jazQ to isolated a total of 73 mutants that enhanced or suppressed its growth and defense parameters. Effort was given to further characterize mutants that suppressed the hindered growth observed in jazQ since these could provide novel mechanistic information on how the activation of JA pathway affect growth processes. Two of these lines carried mutations that impaired JA biosynthesis or signaling: AOS in sjq10 and COI1 in sjq66 . These results demonstrate that the phenotypes observed in jazQ are mainly caused by a positive feedback mechanism that involves the upregulation of genes involved with JA biosynthesis and further degradation o f the remain ing JAZ repressors (Browse 2008; Campos et al. 2014 ; Kazan and Manners 2008 ). In agreement with this hypothesis, we did observe higher expression of genes involved with JA biosynthesis (such as AOS, AOC and OPLC1 ) and enrichment in the GO category ÒJasmonic acid metabolic processÓ in jazQ (Table 3.1) . Quantification of the endogenous levels of JAs in the high order mutant may confirm this hypothesis . The identification of phyB as the causal mutation leading to upregulated growth an d defense in sjq11 was based on the long hypocotyl phenotype observed when this mutant was grown under constant white light (Figure 3.11 ). Interestingly, a mong the 51 sjq lines isolated, only sjq11 showed this phenotype (data not shown) , suggesting that impairment of other genes than PHYB also lead to suppression of the growth phenotype of jazQ. The identification of these 159!mutation s can provide exciting evidence for the involvement of novel components and pathways in controlling the flux o f resource allocation. Phenotypic characterization of the genetically reconstituted sjq11 , jazQ phyB , confirmed that the large rosettes, early flowering phenotype and improved defense parameters observed in sjq11 was caused by the impairment in phyB when i n the jazQ genetic background. One interesting aspect of this finding is the observation that phyB mutant prese nts augmented growth (Figure 3.16 ; Mockler et al. 1999 ), but is severely impacted in defense processes (Figure 3.19 ; Cerrudo et al. 2012; Moreno et al. 2009) . These phenotype s are contrasting as compared to jazQ (less defense, more growth) , indicating that the activation of specific transcriptional circuits can culminate in strikingly different patterns of resource allocation. Surprisingly, results obtained with jazQ phyB suggest that both pathways could be activated in parallel to allow concomitant upregulation of growth and def ense. This hypothesis is corroborated by the phenotype of transgenic 35S:PIF4 jazQ plants , which are upregulated i n defense responses, but show si milar growth as WT (Figures 3.23 and 3.24 ). The observation that 35S:PIF4 plants do not display rosettes as large or flowering as early as jazQ phyB suggest that the expression of other PIF family members , such as PIL1 and REP1 (whose expression is found to be upregulated in jazQ phyB ) may be fundamental to fully suppress the hindered growth in jazQ plants. Uncoupling JA and p hytochrome B transcriptional networks to activate gro wth and defense The JA and phytochrome B tran scriptional circuits evolved as important sensors of environmental signals and their action results in significant changes in plant development. Besides triggering antagonistic physiological responses (growth versus defense), the se two 160!networks operate in a remarkably similar manner (Fig ure 3.25 ): Both pathways are regulated by environmental signals that are capable of inducing molecular changes in transcriptional regulators (JAZ and phy B), releasing a set of basic helix -loop -helix (bHLHs) TFs that bind similar DNA motifs (G -box) to induce developmental responses. In the case of phyB, alterations in R:FR light ratios lead to its dissociation from PIFs, allowing this family of TFs to bind DNA and trigger responses such as organ elongation and cell growth (Che n and Chori 2011, Lorrain et al. 2008). Environmental cues that activate the JA pathway (such as mechanical wounding) promote the degradation of the JAZ repressors , relieving TFs such as MYCs from repression to activate the expression of defense -related ge nes (Browse 2008; Campos et al. 2014). T o correct ly modulate developmental plasticity , these two transcriptional circuits evolved as a binary module, where activation of one is associated to deactivation (repression) of the other. For this purpose , plants utilize different nodes of crosstalk that precisely regulate the status of the module. One of the aforementioned nodes in this crosstalk are the DELLA proteins, which physically associate with the JAZ and PIFs to regulate growth and defense proces ses (Kazan and Manners 2010; Yang et al. 2012) . A different scenario of regulation may occur at the DNA sequence level , where PIFs and MYCs would compete for binding to same gene promoters. Even though these two families of bHLH TFs regulate different sets of responses in the JA -phytochrome B module (growth for PIFs and defense for MYCs), they do bind to the same cis -regulatory elements (the G-boxes) and can act either as activators or repressors of transcription (Yadav et al. 2005; Zhang et al. 2013) . Beca use the G -box element is enriched in promoters of growth -associated/ light responsive genes and also defense -related genes ( Mart™nez -Garcia et al. 2000; van der Burg et al. 2008), it is possible that when growth or defense processes are activated , the TFs a ssociated with it (PIFs or MYCs ) would promote the expression of the genes involved with that response but 161! Figure 3.25. Removal of transcriptional regulators rewires a regulatory network to allow concomitant activation of growth and defense . Phytochrome B (phyB) and JAZ proteins work as transcriptional regulators that inhibit the action of transcription factors associated with growth (e.g. PIFs) and defense (e.g. MYCs). Environmental signals such as red light and herbivory trigger alterations in the molecular status of phyB and JAZ, respectively, releasing the transcription factor association that promote growth or defense. Genetic removal of phyB and five JAZ proteins in jazQ phyB rewire these regulatory networks, promoting the concomitant act ivation of growth and defense and also the activation of processes that are now active only in the jazQ phyB background. 162! concomitantly repress the expression of other genes associated with the opposing process. Indeed, it has been shown, for example, that MYC2 can bind to and negatively regulate the expression of light -responsive genes (Yadav et al. 2005) . Surprisingly, our results with jazQ phyB indicate that the JA -phytochrome B module can be genetically uncoupled. The concomitant removal of multiple transcriptional regulators ( phyB and five JAZ genes) allowed the activation of these two transcriptional circuits in parallel, resulting in activation of both growth and defense in jazQ phyB (Figure 3.25 ). Epistatic relationships between jazQ and phyB are generally consistent with a model in which the MYC and PIF transcriptional programs in jazQ phyB are uncoupled and operate independently of one another. In general, jazQ was epistatic to phyB with respect to defense phenotypes (insect resistance, secondar y metabolism, and JA hypersensitivity) whereas phyB was epistatic to jazQ with respect to growth (rosette diameter, petiole and hypocotyl length, and flowering time) (Table 3.3 ). An interesting exception was rosette biomass; although both jazQ and phyB parental lines have low biomass relative Col -0, the biomass of jazQ phyB is similar to that of Col -0. This finding suggests that the combined transcriptional output of the MYC and PIF transcriptional programs act synergistically to drive biomass accumulat ion through metabolic pathways that are unique to jazQ phyB . In future studies, it will be interesting to analyze the specific components of bio mass in this mutant. Our findings suggest that growth -defense antagonism may not be controlled by limitations on metabolic resources but rather by hard -wired transcriptional regulatory programs that exert control over resource partitioning in response to external signals. At this point it still uncertain if the parallel activation of these two pathways may result in a fitness penalty when these plants are grown in more dynamic conditions such as natural environments. However, it is clear that these two regulatory networks are interesting targets for 163!Table 3.3. Epistatic relationships between jazQ and phyB . RELATIVE TO W ILD TYPE TRAIT jazQ phyB -9 jazQ phyB Petiole length $ % % Hypocotyl length 1 n.a % % Rosette diameter $ % % Rosette biomass $ $ n.a Growth Flowering time Late Early Early Glucosinolate accumulation 2 % $!% Anthocyanin accumulation % $!% Sensitivity to exogenous JA % n.a!% Expression of defense -related genes % $ / n.a!% Defense T. ni resistance % $!% n.a. , not affected relative to WT 1Indicates hypocotyl length of seedlings grown in red light. 2Indicates bulk accumulation of indole and aliphatic glucosinolates. The levels of specific glucosinolates did not always fit this pattern. 164! bioengineering research, in efforts to produce crop cultivars with increased productivity and reduced necessity of pesticide appl ication. Methods Plant material and growth conditions The Columbia ecotype (Col -0) of Arabidopsis thaliana was used as a wild -type (WT) parent for all experiments. Soil -grown plants were maintained at 20 +/ - 1oC under 16 h light/8 h dark photoperiod and 120 µE m-2 s-1 light intensity, unless otherwise noted. For the initial 10 days after seed sowing, trays containing potted plants were covered with a transparent plastic dome to increase humidity. For experiments involving growth of seedlings on agar plates, seeds were surface sterilized for 15 min in a solution containing 50% (v/v) bleach and 0.1% (v/v) Triton X -100, washed 10 times with sterile water and then stratified in dark at 4 oC for 2 days. Seeds were then sown on 0.7% (w/v) agar media containing half -strength Murashige and Skoog (MS; Caisson Labs) salts supplemented with 0.8% (w/v) sucrose. Transfer DNA (T -DNA) insertion mutants used for construction of jazQ were obtained from the Arabidopsis Biological Re search Center (ABRC; The Ohio State University) and are named as follows: jaz1 -SM (jaz1 , JIC -SM.22668), jaz3 -GK (jaz3 , GK -097F09), jaz4 -1 (jaz4 , SALK_141628), jaz9 -GK (jaz9 , GK -265H05) and jaz10-1 (jaz10, SAIL_92_D08). The position of T -DNA insertion withi n each gene is shown diagrammatically in Figure 3.1A. jaz3-GK and jaz9-GK lines were backcrossed to Col -0 to remove unlinked T -DNA insertions. jaz10 -1 was backcrossed to Col -0 to remove a qrt1 -2 mutation present in SAIL lines (McElver et al. 2001). jaz4 -1 and jaz10 -1 mutants were previously described (Jiang et al. 2014; Demianski et al. 2013; Sehr et al. 2010). The jazQ phyB 165!sextuple mutant was obtained from a genetic cross between jazQ and the phyB reference allele phyB -9 (Reed et al. 1993). PCR and qPCR analysis PCR -based genotyping of jazQ and lower -order mutants relied on primer sets flanking T -DNA insertion sites, together with a primer recognizing the border of t he inserted T -DNA ( Table 3.4). Reverse transcription -PCR (RT -PCR) was used to confirm th e presence or absence of JAZ transcripts in WT and jazQ plants ( Figure 3.1B). For this purpose, RNA was extracted from 8 -day -old seedlings grown on MS plates containing 20 µM of MeJA. Frozen tissue was homogenized with a mortar and pestle and RNA was extra cted using an RNeasy kit (Qiagen) followed by on -column DNase (Qiagen) treatment. cDNA was reverse transcribed from 1 # g total RNA with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, ABI). RT -PCR was performed using primers designed to amplify the five JAZ genes and the internal control A CTIN1 (AT2G37620) (Table 3.4 ). RT-PCR reactions were performed with the following conditions: 94 oC for 5 min, followed by 30 cycles of 45 sec at 94 oC for denaturation, 30 sec at 52 oC for annealing and 1. 5 min at 72 oC for elongation. Final elongation step was performed at 72 oC for 10min and completed reactions were maintained at 12 oC. Forty elongation cycles were used to detect JAZ4 transcripts, which accumulate at low le vels in WT plants (Chung et al. 2008). qPCR -based measurement of mRNAs was performed with RNA extracted from WT and jazQ 8-d-old seedlings grown on MS medium not supplemented with JA . RNA extraction and cDNA synthesis was performed as described above. Transcript quantification was eva luated on a 166! Table 3.4. List of PCR primers used in this chapter. TARGET NAME PRIMER SEQUENCE NOTES Genotyping JAZ1 JAZ1_GenFw 5Õ-ACCGAGACACATTCCCGATT -3Õ JAZ1_GenRv 5Õ-CATCAGGCTTGCATGCCATT -3Õ dSpm32_alt 5Õ-ACGAATAAGAGCGTCCATTTTAGAG -3Õ JAZ3 JAZ3_GenFw 5Õ-ACGGTTCCTCTATGCCTCAAGTC -3Õ JAZ3_GenRv 5Õ-GTGGAGTGGTCTAAAGCAACCTTC -3Õ pAC161 -LB1 5Õ-ATAACGCTGCGGACATCTACATT -3Õ JAZ4 JAZ4_GenFw 5Õ-TCAGGAAGACAGAGTGTTCCC -3Õ JAZ4_GenRv 5Õ-TGCGTTTCTCTAAGAACCGAG -3Õ pROK2 -LB3 5Õ-TTGGGTGATGGTTCACGTAG -3Õ JAZ9 JAZ9_GenFw 5Õ-TACCGCATAATCATGGTCGTC -3Õ JAZ9_GenRv 5Õ-TCATGCTCATTGCATTAGTCG -3Õ 35S-rseq1 5Õ-CTTTGAAGACGTGGTTGGAACG -3Õ JAZ10 JAZ10_GenFw 5Õ-ATTTCTCGATCGCCGTCGTAGT -3Õ JAZ10_GenRv 5Õ-GCCAAAGAGCTTTGGTCTTAGAGTG -3Õ pCSA110 -LB4 5Õ-GTCTAAGCGTCAATTTGTTTACACC -3Õ Quantification of transcript levels by qRT -PCR JAZ1 JAZ1_RTFw 5'-ATGTCGAGTTCTATGGAATGTTCTG -3Õ JAZ1_RTRv 5'-TCATATTTCAGCTGCTAAACCGAGCC -3Õ JAZ3 JAZ3_RTFw 5'-ATGGAGAGAGATTTTCTCGGG -3' JAZ3_RTRv 5-'TTAGGTTGCAGAGCTGAGAGAAG -3' JAZ4 JAZ4_RTFw 5'-ATGGAGAGAGATTTTCTCGG -3' JAZ4_RTRv 5'-CAGATGATGAGCTGGAGGAC -3' 40 cycles of elongation in PCR cycle were used to detect a JAZ4 transcript. JAZ9 JAZ9_RTFw 5'-ATGGAAAGAGATTTTCTGGGTTTG -3' JAZ9_RTRv 5Õ-TTATGTAGGAGAAGT AGAAGAGTAATTCA -3Õ JAZ10 JAZ10_RTFw 5'-ATGTCGAAAGCTACCATAGAAC -3' JAZ10_RTRv 5'-GATAGTAAGGAGAT GTTGATACTAATCTCT -3' ACT1 ACT1_RTFw 5Õ-ATGGCTGATGGTGAAGACATTCAA -3Õ ACT1_RTRv 5Õ-TCAGAAGCACTTCCTGTGAACAAT -3Õ qPCR AOS AOS_Fw 5Õ-GGAGAACTCACGATGGGAGCGATT -3Õ As in Attaran et al., 2014. AOS_Rv 5Õ-GCGTCGTGGCTTTCGATAACCAGA -3Õ As in Attaran et al., 2014. CAB3 CAB3_Fw 5Õ-CGGAAAGTGAGCCAAGTTTTATCAG -3Õ As in Attaran et al., 2014. CAB3_Rv 5Õ-AGTCTCAAACCATCACATACAACCT -3Õ As in Attaran et al., 2014. 167! Table 3.4 (contÕd). Quantification of transcript levels by qRT -PCR LHCB2.4 LHCB2.4_Fw 5Õ-GGCCACTTCAGCAATCCAAC -3Õ LHCB2.4_Rv 5Õ-GACGGTACGACGCATGATGA -3Õ MYC2 MYC2_Fw 5Õ-AGAAACTCCAAAT CAAGAACCAGCTC -3Õ MYC2_Rv 5Õ-CCGGTTTAATCGA AGAACACGAAGAC -3Õ PIF4 PIF4_Fw 5Õ-GCCGATGGAGATGTTGAGAT -3Õ PIF4_Rv 5Õ-CCAACCTAGTGGTCCAAACG -3Õ THAS THAS_Fw 5Õ-ATGTACGGGGTCAGCGATTG -3Õ THAS_Rv 5Õ-ATGAACCATCCACCGTTTGC -3Õ TGG2 TGG2_Fw 5Õ-CAGCACAGAAGCTCGCCTTT -3Õ TGG2_Rv 5Õ-GACCAGGGGGTTGACCATTT -3Õ Identificaton of causal mutations in sjq s AOS AOS_F1 5Õ-CAAAATATGGATACGGGACA -3Õ As in Niu et al. 2011 AOS_F3 5Õ-AAAACTAGTATGGCT TCTATTTCAACCCCT -3Õ AOS_F4 5Õ-CTTCCTCCTCAAGTCATCTCG -3Õ AOS_R3 5Õ-AAAACTAGTCTAAAAG CTAGCTTTCCTTAACG -3Õ AOS_R4 5Õ-CGTAGAAAGCTCGAGCCAAG -3Õ COI1 COI1 -f3 5Õ-ATGGAGGATCCTGATATCAAG -3Õ COI1 -f5 5Õ-GTAGCTGAGATCTGACCACTGCAA -3Õ COI1 -fseq1 5Õ-AGCATCGTTACACACTGCAGGA -3Õ COI1 -rseq1 5Õ-TTGCATTCATATCCCTTATCTCC -3Õ COI1 -r2 5Õ-ATTGCTCGCTCACTGAAGCAAC -3Õ COI1 -r5 5Õ-GCTCTCAGAAGTCAACACCATGGA -3Õ PHYB PHYBgenFw 5Õ GAAGAAACCAAACTTGTATAGTACG -3Õ PHYBgenRv 5Õ AATTTCAACTTTTTGGACGG -3Õ 168! 7500 Fast Real -Time PCR system (Applied Biosciences) using the protocol described by Attaran et al. (2014). Primers utilized for experiments are listed in Table 3.4 . Root growth assays The effect of exogenous JA on seedling root growth inhibition was determined as pr eviously described (Shyu et al. 2012). Seedlings were grown on square Petri plates (F isher) containing MS medium supplemented with the indicated concentration of methyl -JA (MeJA; Sigma -Aldrich). Plates were incubated vertically in a growth chamber maintained at 21 oC under continuous lig ht for 8 d. The p rimary root length was measured with the use of ImageJ software (http://imagej.nih.gov/ij/). WT and mutant lines were grown on the same plate to control for plate -to-plate variation. Quantifi cation of secondary metabolites Anthocyanins we re quantified as described by Spitzer -Rimon et al. (2010), with minor modifications. Petioles were excised from 4 -week-old plants and extracted in 1 ml methanol containing 1% (v/v) HCl. Samples were incubated overnight at 4¡C with constant agitation. Antho cyanin pigments in the resulting extract were measured spectrophotometrically and calculated as A 530 - 0.25(A 657).g-1 fresh weight. Glucosinolates were quantified as described by Barth and Jander (2006) with minor modifications. Eight -day -old seedlings gro wn on MS plates in the absence of MeJA were collected into two -mL tubes (approximately 50 seedlings per tube) and immediately frozen in liquid nitrogen. WT and mutant lines were grown on the same plate to control for plate -to-plate variation. Frozen tissue was lyophilized, ground to a fine powder and extracted with 1 mL of 80% MeOH containing an internal standard (25 nmol sinigrin Sigma - 169!Aldrich). Samples were briefly vortexed, incubated at 75 oC for 15 min, and then centrifuged at room temperature at 10 ,000 x g for 10 min. Resulting supernatants were applied to Sephadex A -25 columns (Amersham). De sulfoglucosinolates were eluted with a solution containing 30 µL of sulfatase (3.0mg mL-1; Sigma) and 70 µL water (HPLC -graded). Following an overnight incubation in the dark at room termperature, 200 µL 80% MeOH and 200 µL water were added to each sample. Samples were then lyophilized for 2 h and dissolved in 100 µL water. Desulfoglucosinoaltes were detected by HPLC and quantified as described (Barth and Jander 2006). For each independent experiment, ten biological replicates per genotype were used fo r quantification of anthocyanin and glucosinolates levels. Insect feeding assays Insect feeding assays were performed with soil -grown plants maintained in a growt h chamber at 19oC and a photoperiod of 8 hrs light (120 µM.m -2.s-1)/16 hrs dark. Neonate Trichoplusia ni larvae (Benzon Research) were transferred to the center of fully expanded rosette leaves of six -week-old plants, as pre viously described (Herde et al. 2013). Four larvae were reared on each of 12 plants per genotype. Plants were then covered with a transparent dome and returned to the chamber for ~10 days, after which larval weights was measured. Rosett e phenotypes and flowering time Rosette and flowering phenotype characterization was performed with three to four week -old soil -grown plants using at least 10 plants per measurement, unless indicated otherwise. Petiole length of the excised 3 rd true leaf was measured with a calip er. At this time stage in development, the total number of true leaves on each rosette was counted to assess the developmental status of 170!each genotype. Bolting time was measured in a separate set of plants by scoring the number of true leaves on the main stem and the numbe r of days from sowing until bolting (i.e., flower buds visible in the center of the rosette). The same set of plants was subsequently used to measure the length of time to opening of the first flower. Rosette diameter and projected leaf area were determine d by photographing rosettes with a Nikon D80 camera . The resulting images were used to calculate Feret diameter using ImageJ (http://imagej.nih.gov/ij/) and total area leaf using GIMP software (http://www.gimp.org). Leaf dry weight was determined by weighi ng the excised rosette (without roots) after freeze drying for two days in a lyophylizer. jazQ mutagenesis experiment and identification of causal mutations in sjq plants Approximately 50,000 jazQ seeds were mutagenized by immersion in a solution of 0.1% or 0.2% (v/v) ethyl methanesulfonate (EMS, Sigma -Aldrich) for 16 hr at room temperat ure, with constant agitation. Se eds (M1 generation) were thoroughly washed with H 2O, stratified in dark at 4 oC for two days and the n immediately sown on soil. M2 seed was obtained from 16 pools of self -pollinated M 1 plants (approximately 1,000 M 1 plants/pool). Soil -grown M 2 plants (~2000 plants/pool) were visually screened for individuals in which one or more jazQ phenotypes, including compact rosette size, delaye d flowering time and anthocyanin accumulation were altered. Putative sjq (suppressors of the jazQ ) and ejq (enhancers of the jazQ ) mutants were rescreened in the M 3 generation to confirm heritability of phenotypes. Identification of causal mutations in sjq10 and sjq66 was based on JA -phenotypes observed for plants grown in soil and MS plates containing 25 µM MeJA. PCR p rimers for amplification AOS and COI1 genes in sjq10 and sjq66 , respectively, are described in Table 3.4 . Insight into the causal mutation in sjq11 was obtained from hypocotyl elongation assays 171!performed with monochromatic light as described by Fankhouser and Casal (2004) , with minor modifications. WT, jazQ and sjq11 (M3 generation) seeds were plated on MS medium lacking sucr ose and stratified at 4 oC in dark for two days. To improve synchronous seed germination, a 3 hr pulse of white light was administered. Plates were then returned to darkness for one day at 21oC and then transferred to monochromatic LED chambers outfitted to emit blue (470 ± 20 nm Ð 5 µE), red (670 ± 20 nm Ð 25 µE) or far -red (740 ± 20 nm Ð 5 µE) light. As controls, the light -sensing mutants phyA -75, phyB -9 and cry1-400 were included (gift from Dr. Rob Larkin, Michigan State University, see Reed et al. 1993; Ruckle et al. 2007) and a set of plates containing each genotype was maintained in darkness. WT and mutant lines were grown on the same plate to control for plate -to-plate variation. Following three days of grown under the specified mono chromatic light condition , seedling hypocotyls were scanned for length measurement using the ImageJ software. Allelism tests were performed with F 1 plants obtained from the cross between sjq11 and phyB -9. Seedlings were grown on MS medium plates and incu bated horizontally in a growth chamber maintained at 21 oC under continuous light for 3 d. Global g ene expression profiling (RNA -seq) Global gene expression profiling in 8 -day -old whole seedlings was assessed by mRNA sequencing (RNA -seq) performed on the Ilumina HiSeq 2000 platform. Seedlings were grown on solid MS medium supplemented with sucrose but not JA, and harvested for RNA extrac tion (~200 seedlings per RNA extraction). WT and mutant seedlings were grown on the same plate to minimize plate -to-plate variation. Two independent RNA -seq experiments were performed. The first experiment compared expression profiles of WT and jazQ only, whereas the second 172!experiment compared transcript abundances in WT , jazQ , phyB -9 and jazQphyB . In both experiments, three independent RNA samples (biological replicat es) were sequenced per genotype. Total RNA was isolated as described above and RNA integri ty was assessed on a 2100 Bioanalyzer (Agilent Technologies). All samples used for sequencing had an integrity score of at least 7.0. Single -end (50 bp) sequencing was performed at the Michigan State University Research Technologies Service Facility (htt ps://rtsf.natsci.msu.edu). Barcoded sequencing libraries were constructed using the Illumina RNAseq kit according to the manufacturerÕs instructions . Samples were multiplexed in six libraries per lane. The average number of sequencing reads were 22.1 ± 1.6 and 18.42 ± 4.3 million reads per sample in the first and second experiment, respectively. Raw sequencing reads were assessed with Illumina quality control tools filters and FASTX toolkit (http://hannonlab.cshl.edu /fastx_toolkit/). Reads were mapped to g ene models in the TAIR10 with the program RSEM (version 1.2.11) set for d efault parameters (Li and Dewey 2011). D ata was expressed as transcripts per million reads (TPM; Wagner et al. 2012). DESeq, (version 1.18.0; Anders and Huber 2010) was used to assess differential gene expression by comparing TPM values in WT to that in the mutan t line (Figure 3.26 ). Gene onthology (GO) analysis of enriched functional categ ories was performed using BinGO (version 2.44, Maere et al. 2005). The default Benjamini & Hochberg multiple testing correction was used to calculate over - and underrepresented GO categories among differentially expressed genes, using a P value of <0.05. The same RNA utilized for high throughput sequencing was used in experiments to validate seq uencing data by qPCR (Figure 3.27 ), as described in Attaran et al. (2014). Primers utilized for experiments ar e listed in Table 3.4 . 173! Figure 3.26. Comparison of gene expression profiles between wild type (WT) and various mutants analyzed in this study. Scatter plots of expected counts reads obtained by full transcriptome sequencing (RNA -seq) for WT versus the mutants studied in this work: (A) WT versus jazQ, experiment 1, (B) WT versus jazQ , experiment 2 (see methods), (C) WT versus phyB -9 and (D) WT versus jazQ phyB . Red dots indicate genes called as differentially expressed in the mutants according to DESeq statistical package ( p-value<0.05). 174! Figure 3.27. Validation of RNA -seq data by qPCR. Samples submitted for RNA -seq analysis were evalua ted for the expression of specific genes by qPCR. Values represent the fold change in expression of jazQ over WT measured by qPCR (y -axis) or RNA -seq (x -axis). 175! Overexpression of PIF4 in the jazQ background The 35S::PIF4 -TAP overexpression construct was kindly provided by Dr. Michael Thomashow and is previous ly described (Lee and Thomashow 2012). Transformation of WT and jazQ plants with Agrobacterium tumefaciens (strain C58C1) was performed using the flo wer dip method (Clough and Bent 1998). Multiple independent transformed lines (T1 generation) were selected on MS plates containing gentamycin and transferred to soil for subsequent analysis. Homozygous lines were selected by testing the T3 progeny for gentamycin resistance. 176! Acknowledgements We than k Dr. Yuki Yoshida for development of the jazQ and jazQ phyB mutant s and characterization of sjq11 under monochromatic light , Dalton Oliveira for help with the suppressor screen , Alexandra Lantz and Kayla Moses for characterization of suppressor mutants and Dr. Ian Major for assistance with analysis of RNA -seq data. We also thank the Brazilian National Council for Scientific and Technological Development (CNPq) for a fellowship to Dalton Oliveira. T his work was supported by the National Institutes of Heal th (Grant no. 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Summary of dissertation From its first isolation in the early 1960Õs (Demole et al. 1962) to the recent crystallization of its co-receptor complex (Sheard et al. 2010), scientific interest in jasmonates (JAs) has made a transition from being a simple constituent of the jasmine flower scent to a ubiquitously occurring hormones that play s essential roles in pl ant development and immune function . The identification of the JASMONATE ZIM -domain (JAZ) family of proteins marked a major step towards understanding JA signaling (Chini et al. 2007; Thines et al. 2007 , Yan et al. 2007 ), but , at the time this dissertation research started, little was understood about the molecular mechanisms of JAZ function. Functional redundancy among the JAZ family members hindered research to elucidate the biological relevance of these repressors in plant g rowth and defense. In this dissertation, I first desc ribe how alternative splicing of a specific Arabidopsis thaliana JAZ gene, JAZ10 , produces stabiliz ed splice variants that function to attenuate JA responses upon induction . This function is dependent on an intron -retention event that truncates the C -terminal end of the degron, thus impairing interaction of the splice variant isoform with COI1. Because this mechanism of protein stabilization is distinct from that of other JAZ repressors (e.g. JAZ8, Shyu e t a l. 2012), t hese findings suggest a degree of functional specificity among the JAZ family members. This dissertation research also employed the higher order jazQ mutant to address the function of JAZ genes. I showed how constitutive activation of the JA signaling in this mutant leads to a shift in the allocation of resources between growth and defense processes . Screening of an EMS -mutagenized population of jazQ plants lead to the identification of a phyB mutant ion that suppresses the slow -growth phenotype of jazQ without significantly altering the enhanced defense phenotype. Characterization of jazQ phyB plants indicated that a genetic uncoupling of 188!the JA -phyB binary module trigger s the concomitant activati on of growth and defense processes. Taken together, m y findings suggest that the JAZ proteins are essential modulators of plant growth and defense. Alternative splicing of JAZ transcripts expand s the spectrum of repressors that participate in the JA -signa ling path way. It was previously shown that JAZ10 is subjected to alternative splicing, producing three protein isoforms (JAZ10.1, JAZ10.3 and JAZ10.4) that differentially interact with CORONATINE INSENSITIVE1 (COI1) in the presenc e of JA -Ile (Chung and Howe 2009; Chung et al. 2010 ). However, the biological function of these variants was still unknown. In Chapter Two of this dissertation , I describe the func tional characterization of the JAZ10 splice variants and show that, upon induction by mechanical wo unding, the JA -labile isoform JAZ10.1 is quickly removed from c ells. In contrast , the stable isoforms JAZ10.3 and JAZ10.4 accumulate and are retained for longer periods of time in JA -elicited cells . In a series of complementation assays utilizing the jaz10 -1 null mutant, which is insensitive to exogenous JA (Demianski et al. 2012), I demonstrate d that JAZ10.3 and JAZ10.4 but not JAZ10.1 function to attenuate various JA responses , including inhibition of root elongation and expression of JA -responsive genes. The observation that JAZ8 cannot complement the ability of stable JAZ10 splice variants to attenuate JA responses point to a unique role of JAZ10 in the regulation of JA signaling . Furthermore, t hese results suggest that alternative splicing can lead to p roduction of a range of JAZ variants that , based on differences in stability, perform different functions. I then focused my studies on understanding the mechanism by which the most abundant of the JAZ10 protein isoforms in JA -elicited cells, namely JAZ10.3, acts as a potent repressor . JAZ10.3 lacks the seven C-terminal amino acids of the Jas motif, an alteration that impairs its interaction with COI1 in the presence of JA -Ile . I found that addition of a single amino acid to 189!the C -terminus of JAZ10.3 (to generate JAZ10.3+L Jas21 ) is sufficient to fully restore ligand -dependent interaction with COI1. Interestingly, this affected region of the Jas motif corresponds to the C -terminal end of an alpha -helical region that was proposed to dock the JAZ substra te to COI1 in a manner that facilitates JA -Ile dependent formation of the COI1 -JAZ co -receptor complex (Sheard et al. 2010). My results therefore suggest that the intron -retention event that gives raise to JAZ10.3 impedes COI1 -JAZ interaction by disrupting the integrity of this alpha -helix. Furthermore, t he observation that the intron -retention event is conserved among the majority of JAZ genes in Arabidopsis and likely JAZ genes in other plants as well indicates an evolutionary importance for these type of events . In Chapter Three of the dissertation, I describe a strategy to overcome the functional redundancy in the JAZ family by genetic removal of multiple JAZ genes. C onstruction of the jazQ mutation resulted, for the first time, in constitutive activati on of JA responses and upregulation of defense processes. As described by the growth versus defense paradigm (Herms and Mattson 1992; Huot et al. 2014), jazQ is also hindered in the growth of above - and below -ground organs . Transcriptional, metabolic and m orphological characterization of jazQ illustrates how the constitutive activation of the JA pathway shifts the growth -defense equilibrium to wards defense processes and highlights the significance of this hormone and the JAZ proteins as regulators of resource allocation. The phenotypes of jazQ provide a powerful tool to identify regulatory genes that control growth -defense tradeoffs . I isolated an EMS -mutagenized supressor line (sjq11 ) that retains defense -related phenotypes (e.g. anthocyanin accumulation) of jazQ but exhibits a robust growth phenotype comparable to WT plants. I identified the genetic basis of the sjq11 phenotype, which was tracked to a non -sense mutation that in PHYB. Genetic reconstitution of sjq11 through 190!construction of the jazQ phyB sextuple mutant confirmed that this combination of mutations results in uncoupling of growth -defense antagonism. These results were surprising because phyB mutants are known to exh ibit increased extension -type growth (e.g. petiole elongation) and impaired defense against insect s and pathogen s (Cerrudo et al. 2012; Moreno et al. 2009). Moreover, current models i ndicate that the JA -phyB crosstalk is a binary module that perceives external signals and modulate resource allocation in response t o changing environmental conditions (Ballar” 2014; Kazan and Manners 2012; Leone et al, 2014; Moreno et al. 2009). In this context, activation of one branch of th is module is typically associated with repression of the counterpart branch . However, I found that the combination of the jazQ and phyB mutations simultaneous increases both growth and defense. This uncoupling of growth -defense antagonism results from a tr anscriptional reprogramming that allows not only the simultaneous expression of defense - and growth -related genes, but also the activation of new transcriptional circuits that are not active in either jazQ and phyB parental genotypes. My findings suggest t hat growth -defense antagonism may not be dictated by constraints on metabolic resources but rather by hard -wired regulatory programs that exert control over resource partitioning in dynamic environments. To summarize, research in this dissertation demons trate s how plants utilize the JAZ proteins to regulate growth and defense processes upon environmental fluctuations (Figure 4.1) . In the absence of stress (resting state), JAZ proteins interact with and inhibit the action of MYC2 transcription factors ( TFs) associat ed with defense responses . JAZ also interact with negative regulators of growth (DELLAs), thereby allowing PIF TFs to regulate the expressi on of growth -associated genes (Yang et al. 2012). In this resting state, resources are spent primarily on growth processes. However, u nder conditions of environmental stress such as mechanical wounding or insect herbivory, a ccumulation of bioactive JA (JA -Ile) promotes the formation of the COI 1-JA- 191! Figure 4.1. JAZ pr oteins are modulators of plant growth and defense. In absence of stress (Resting state), JAZ proteins act as growth promoters, hindering the action of defense -related transcription factors (TFs) (MYCs) and negative regulators of growth (DELLAs). TFs assoc iated with growth processes (PIFs) are free to bind to and activate trans - 192!Figure 4.1 (contÕd) . cription of growth -related genes, but also to repress the expression of defense -related genes. In this resting condition, plants prioritize the allocation of re sources to growth. However, stressful situations such as insect herbivory trigger a burst of JA -Ile, which allows the formation of COI1 -JAZ complexes that promote JAZ degradation. In this condition (Active state), MYCs are freed from repression and activat e defense -related genes. Removal of JAZ also promotes DELLA interaction with PIFs as a further mechanism to repress growth. In this active state, plants allocate their resources mainly to defense. Stress alleviation leads to accumulation of JA -stable JAZ p roteins (e.g. JAZ10.3), which interact with MYCs TFs and DELLAs even in the presence of high JA -Ile levels. In this condition (Attenuation state), defense -related processes are attenuated and growth is resumed. Finally, upon stress removal (relaxation), JA -stable JAZ proteins are removed by an unknown mechanism, allowing the system to be JA -sensitized again if necessary. 193! JAZ complex that targets the JAZ proteins from degradation . JAZ removal allows the MYC family of TFs to bind to JA -related genes and a ctivate defense responses, and also inhibit growth -related gene expression (Yadav et al. 2005) . Removal of JAZ also promote s DELLA -PIF inter action as another proposed mechanism of JA -mediated growth suppression. In this scenario, resources are invested mainly i n defense processes. A s part of a negative feedback loop to attenuate JA responses, de novo synthesis of stable JAZ such as JAZ10.3 serves to inhibit the action of defense -related TFs even in the presence of JA -Ile. This attenuation of JA responses presumably balances growth -defense tradeoffs and also permits return to resting state when stress conditions subside . Future perspectives In the past few years, numerous discoveries have advanced our understanding of JA biology and how this hormone evol ved as a mechanism to maximize plant fitness in an ever -changing environment . The JAZ proteins were discovered less than 10 years ago as the missing link in the JA signaling pathway (Chini et al. 2007; Thines et al. 2007, Yan et al. 2007) . (+ )-7-iso-JA-Ile was identified as the bioactive form of the hormone (Fonseca et al. 2009 ; Sheard et al. 2010; Staswick and Tiryaki 2004 ) and biochemical pathways for catabolism of this bioactive molecule are now understood (Heitz et al. 2012; Koo et al. 2011 ; Koo a nd Howe 2012; Koo et al. 2014 ). The crystal structure of the COI 1-JAZ JA -Ile co -receptor complex was resolved, unraveling the mechanism of JA -Ile perception (Shea rd et al., 2010). A rapid ly expanding list of TFs and co -repressors involved with JA -signaling is providing additional insight into how JA responses are coordinated (Fern⁄ndez -Calvo et al. 2010; Nakata et al. 2013; Pauwels et al. 2010; Qi et al. 2011; Song et al. 2011 ; Song et al. 2013; Yang et al. 2012 ). Several nodes for crosstalk between the JA 194!and other signaling pathway s have been identified (Campos et al. 2009; Grunewald et al. 2009; Lorenzo et al. 2002; Moreno et al. 2009; Song et al. 2014 ; Thaler et al. 2012; Yang et al. 2012 ) and there is now a firm mechanistic understanding of JA-triggered plan t immunity ( Howe and Jander 2009; Campos et al. 2014). While these discoveries are helping us to understand the broad biological relevance of JA , they also raise new and exciting questions. Recent studies have identified several JAZ p roteins that interact weakly or not at all with COI1 (Chung et al. 2010; Moreno et al. 2014; Shyu et al. 2012; Thireault et al. 2015). The mechanism s involved in turnover of these stable JAZ proteins , which include JAZ10.3, JAZ10.4, JAZ8 and JAZ13, remain to be determined . Such mechanisms presumably exist in order for cells to recover from JA -induced stress and to become Òre -sensitizedÓ to the hormone ( Figure 4.1). One hypothesis is that a ligand other than JA -Ile can promote COI1 int eraction with these proteins. Hy droxylated and carboxylated forms of JA -Ile that are synthesized in response to wounding have little or no capacity to promote the formation of COI1 -JAZ complexes (Heit z et al. 2012; Koo et al. 2011). However, it remains to be tested whether these JA-Ile derivatives can promo te COI1 interaction with the stable JAZ repressors referred above . This function can also be performed by different endogenously found JA -amino acid conjugates (other than JA -Ile), which have been shown to promote COI1 -JAZ inte raction in vitro (Katsir et al. 2008). The se alternative s suggest that the JA -stable JAZ form a different binding pocked that allows the utilization of a different com pound other than JA -Ile as the molecular glue to promote the formation of COI1 -JAZ comple xes. Identification of novel JAZ genes and splice variants may greatly expand our understanding o f the biology of JA. Recent homology studies with JAZ8 lead to the description a new JAZ gene in Arabidopsis , called JAZ13 . JAZ13 possesses unique features not described for 195!other JAZ family members (Thireault et al. 2015). One example is a C -termi nal Ser -rich tail that is a likely site for protein phosphorylation , suggesting a new mechanism to regulate JAZ function. The increasing availability of genome -scale datasets is facilitating the identification of new JAZ genes and JAZ splice variants in diverse species (Chung et al. 2010; Hong et al., 2014; Ye et al. 2009) . Molecular characterization of these genes promises to i mprove our comprehension of the versatility of JA as a regulator of plant growth and development . An elegant recent example was demonstrated in rubber tree ( Hevea brasiliensis ) where characterization of the JAZ gene family provided new insight into the role of JA signaling in wound -induced latex production (Hong et al. 2014) . In Chapter Three of this dissertation , I described the JA -phyB transcriptional network as a binary module that is rewired upon recognition of specific external signals to modulate the tradeoffs between growth and defense. Similar regulatory modules involving other signaling pathways possibly exist to control the allocation of finite resources in the face of changing environmental conditions . In this sense, t he m olecular characterization of crosstalk nodes between JAs and other hormone response will allow us to better understand the regulatory circuits involved in plant growth and defense . Analysis of crosstalk between JA and growth -related hormones such as auxin, gibberellin and brassinosteroids are already leading to the identification of signaling components that are essential for proper tuning of plant development (Campos et al. 2009; Grunewald et al. 2009; Yang et al. 2012 ). It i s tempting to speculate that some of the jazQ suppressor mutants identified during the course of this dissertation research (Chapter Three) are indeed affected in these networks . Further characterization and identification of the causal suppressor mutation s is needed to test this hypothesis. 196! Experimental designs that more accurately reflect the dynamic condition in natural environment s will ultimately be needed to discern the complexity of regulatory circuits involved in resource allocation. Phenotypic cha racterization of jazQ phyB and related mutants in a broad range of environmental conditions (e.g. fluctuating temperature and light ), including field studies will likely provide valuable insight into this question . Empirical evidences obtained from these types of experiments may indicate, for example, how plants prioritize specific developmental programs in the face of multiple stress es (Moreno et al. 2009). It is known that natural population s of herbivores dictate polymorphisms in defense traits (Zt et al. 2012) and that competition with naturally occurring , fast growing plants can trigger a shift in the growth -defense equilibrium (Agrawal et al. 2012) . This ecological perspective provides a plausible strategy to bridge basic and applied science , which may lead to the development of crop s that are optimized in growth and defense processes . The development of new technological tool s and current pace of discovery mark a new era in understanding the biological function of JA in resource allocation. Interdisciplinary studies that combine large -scale -omics analysis, synthetic biology, mathematical modeling and system s biology will aid in the quest to untangle the intricate web of regulatory circuits in which JA participates to maximize plant fitness i n response to myr iad of environmental conditions . In conclusion, there is no better time to be studying JA biology. 197! 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